Lactobacillus biotransformation process

Abstract

A method for producing cellular metabolites by cultivating lactobacillus cells in cell culture, comprising feeding the cell culture in an aerobic reactor system during a production phase with a feed medium comprising a carbon source for bioconversion into cellular metabolites, and isolating the cellular metabolites from the cell culture, and the lactobacillus strains deposited as DSM 33056, DSM 33057, DSM 33058, DSM 33059 and DSM 33060, or progeny or derivatives of any of the foregoing strains.

Description

Technical Field

[0001] The present invention relates to a method for culturing lactobacillus diolivorans to produce cell metabolites, and a novel lactobacillus strain. Background Art

[0002] The chemical modification of small molecules by microorganisms is often referred to as biotransformation or biosynthesis. Biotransformations are widely studied for the production of materials for industrial use. The microbial production of alcohols, sugars or organic acids is a promising approach to obtain end products or building-block chemicals from renewable carbon sources (e.g. monomers for polymer synthesis). The most common chemicals produced by biotransformation processes are 1,3-propanediol, citric acid, lactic acid or succinic acid, and sugar alcohols such as mannitol. The biotechnological conversion of glycerol to 1,3-propanediol is usually carried out by bacteria under anaerobic conditions.

[0003] Willke et al. (Eur. J. Lipid Sci. Technol. 2008, 110, 831-840) reviewed the bioconversion of glycerol to 1,3-propylene glycol without using fossil resources. Microorganisms of the Clostridiaceae, Enterobacteriaceae, and Lactobacillus genera are reported to be promising candidates for the industrial production of 1,3-propylene glycol.

[0004] Although microbial processes for producing valuable chemicals from renewable resources using microorganisms are known, most production organisms rely on one or a few carbon sources, and most known production organisms require high-purity carbon sources. Therefore, the carbon source is a major cost factor.

[0005] WO 2013064682 A2 discloses lactobacilli used in a bioconversion process, wherein the raw material used in the bioconversion process is a complex mixture of organic carbon sources containing carbohydrates to be bioconverted, such as crude glycerol, of less than industrial purity. In all cases, nitrogen is used to provide anaerobic conditions in the reactor during cell culture.

[0006] It would be desirable to reduce the cost of producing cellular metabolites in Lactobacillus (L. diolivorans) cell cultures.

[0007] Lactobacilli are widely used in the food and feed industries. WO 2010 / 122165 A1 relates to a method for producing sourdough and baked goods with an extended shelf life by co-fermentation of selected lactobacilli (e.g., L. buchneri, L. parabuchneri, and L. diolivorans) with biopreservation.

[0008] Lactobacillus diolivorans sp. nov. (DSMZ 14421, LMG 19667) was isolated from aerobic stabilized corn silage and identified as a 1,2-propylene glycol-degrading bacterium (Krooneman et al. International Journal of Systematic and Evolutionary Microbiology 2002, 52, 639-646).

[0009] WO 2006 / 007395 A1 and WO 2007 / 103032 A2 describe bacterial additives to (sugar cane) silage using inocula comprising lactobacilli, in particular in order to reduce the dry matter content of the silage.

[0010] Vivek et al. (Bioresource Technology 2016, 213:222-230) described the valorization of pure and crude glycerol to 1,3-propanediol using a Lactobacillus brevis isolate under anaerobic conditions.

[0011] Jolly et al. (Journal of Bioscience and Bioengineering 2014, 1182: 188-194) disclosed the biosynthesis of 1,3-propanediol from glycerol using Lactobacillus reuteri under anaerobic conditions (nitrogen purge). Summary of the Invention

[0012] The object of the present invention is to provide a bioconversion process suitable for producing cell metabolites in lactobacillus cell cultures, which process is simple and inexpensive, and in particular obtains cell metabolites in high yield.

[0013] Said objects are solved by the claimed subject matter and are further elaborated herein.

[0014] The present invention provides a method for producing cellular metabolites by culturing lactobacillus cells in cell culture, comprising feeding the cell culture in an aerobic reactor system using a feed medium containing a carbon source for bioconversion into cellular metabolites during a production phase, and isolating the cellular metabolites from the cell culture.

[0015] According to one specific aspect, the aerobic reactor system does not include or utilize any device or method capable of protecting the cell culture from oxygen supply. Specifically, the aerobic reactor system does not include or utilize means for purging (or supplying or otherwise actively introducing) nitrogen or any anaerobic gas (particularly gases other than air or 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 methods described herein have been shown to successfully produce bioconversion products in high yields without requiring any device that limits the oxygen content of the cell culture.

[0016] Specifically, 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 less than 0.1 (vol / vol / min).

[0017] It has been demonstrated that the Lactobacillus cell cultures used in the methods described herein successfully produce cellular metabolites by bioconverting a carbon source, despite the absence of oxygen-limiting devices in the reactor system. Although the prior art describes the use of organisms protected from oxygen during the production phase (under strictly anaerobic conditions), it has been found that the cell cultures described herein readily consume any oxygen present in the cell culture during the production phase, thereby generating anaerobes in situ without any external measures to limit oxygen and being surprisingly insensitive to any such oxygen present in the aerobic reactor system described herein, while still being able to produce bioconversion products in high yields.

[0018] Specifically, 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 (as determined by a standard assay, such as an assay employing luminescence quenching, e.g., using a VisiFerm RT-PCR assay, according to the manufacturer's instructions). TM OD Sensor HAMILTON). In an exemplary standard method, the luminescence of certain organic pigments (luminophores) is quenched in the presence of oxygen. The luminophores absorb 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 luminophore to the oxygen. The luminophores no longer emit fluorescent light, and the measurable fluorescence signal decreases.

[0019] Specifically, the dissolved oxygen is in the range of 0.1-5%, preferably 0.1-1%.

[0020] According to a specific aspect, during the production phase, the cell culture is maintained at a pH in the range of 4 to 7 and a temperature in the range of 25 to 40° C. Preferably, the pH is about 5.7 (“about” is understood as + / - 0.1) and the temperature is about 30° C. (“about” is specifically understood as + / - 1°).

[0021] According to a specific 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.

[0022] According to a specific aspect, the carbon source contained in the feed medium includes one or more carbohydrates selected from 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 the D- and N- forms of the sugars can be used; in addition, the sugars can also be derivatized, for example, by acetylation. Preferred sugar acids are uronic acids, such as glucuronic acid, galacturonic acid, or derivatives of such acids, such as methyl glucuronic acid.

[0023] According to a specific aspect, the carbon source comprised in the feed medium is selected from the group consisting of glycerol, crude glycerol, glucose, fructose, lactose, sucrose, starch, biomass hydrolysate, cellulose, lignocellulose, 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 a carbohydrate having a purity lower than technical grade and an ash content of at least 0.1% (w / w).

[0024] The methods described herein specifically utilize a raw material as a carbon source that includes a complex mixture of an organic carbon source including one or more carbohydrates to be bioconverted and impurities, such as one or more carbohydrates having a purity lower than industrial grade.

[0025] In particular, the raw 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).

[0026] In a specific embodiment, raw material refers to a material selected from the group consisting of unprocessed glycerol (also referred to herein as crude glycerol), sugar cane, sugar beet, starchy plants, cellulose, hemicellulose, lignocellulose (including lignocellulose plant material or lignocellulose hydrolysate) and chitin (e.g., chitin-containing material from shellfish). Unprocessed glycerol can be obtained from fatty acid production, biodiesel production, bioethanol production or soap production. Other raw materials can come from waste streams of the paper industry, sugar industry or timber industry.

[0027] In particular, the carbon source is crude glycerol and is used in a concentration of up to 90% (w / w) in the feed medium, in particular at least any 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%.

[0028] In particular, the crude 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).

[0029] In particular, crude glycerol is obtained as a by-product of fatty acid production, soap production, bioethanol production or biodiesel production.

[0030] The methods described herein specifically produce target cellular metabolites. In particular, the cellular metabolites are not constitutively produced (or primary metabolites), i.e., supporting bacterial growth, but are secondary metabolites or such metabolites produced biosynthetically by adding a carbon source to be bioconverted during the culture.

[0031] Specifically, cellular metabolites are chemical substances or low molecular weight organic molecules (small molecules).

[0032] Specifically, the cellular metabolites are 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-hydroxypropionaldehyde, 3-hydroxypropionic acid, butyric acid, valeric acid, hexanoic acid, adipic acid, succinic acid, fumaric acid, malic acid, 2,5-furandicarboxylic acid, aspartic acid, Acid, saccharic acid, gluconic acid, glutamic acid, itaconic acid, levulinic acid, acrylic acid, propanol, isopropanol, 1-butanol, 2-butanol, pentanol, hexanol, heptanol, octanol, butylene glycol, 2,3-butanediol, 3-hydroxybutyrolactone, xylitol, arabitol, sorbitol, mannitol, vitamin C, riboflavin, thiamine, tocopherol, cobalamin, pantothenic acid, biotin, pyridoxine, niacin, folic acid, 3-hydroxybutyrolactone, diaminohexane, and dihydroxyacetone.

[0033] Specifically, the chemical is an organic acid or alcohol; exemplary chemicals produced are 1,3-propanediol, lactic acid, 3-hydroxypropionic acid, and mannitol.

[0034] Specifically, glycerol (especially crude glycerol) is bioconverted to produce at least one or more of 1,3-propanediol, 3-hydroxypropionaldehyde and / or 3-hydroxypropionic acid.

[0035] A specific embodiment relates to a method wherein

[0036] (i) the production of 1,3-propylene glycol from crude glycerol; or

[0037] (ii) producing 3-hydroxypropionic acid from crude glycerol; or

[0038] (iii) producing 3-hydroxypropionaldehyde from crude glycerol; or

[0039] (iv) producing lactic acid from lignocellulose or lignocellulosic biomass hydrolysate; or

[0040] (v) Production of mannitol from sugar beet extract or hydrolysate.

[0041] According to a specific aspect, feed medium is continuously added to the cell culture during the production phase.

[0042] According to a specific aspect, the production phase is performed in fed-batch mode or in continuous mode.

[0043] Specifically, prior to the production phase, cell culture is performed in batch mode using a growth medium to accumulate biomass.

[0044] Specifically, the growth medium that allows biomass accumulation comprises a carbon source, a nitrogen source, a sulfur source and a phosphate source. Typically, this medium also comprises trace elements, vitamins and amino acids.

[0045] Typically, the growth medium comprises more than one carbon source to obtain a high concentration of biomass, eg, at least 5 g / L, preferably at least 7.5 g / L, even more preferably at least 10 g / L of biomass.

[0046] In particular, the growth medium comprises a carbon source selected from hexoses, pentoses or heptoses. Specifically, the carbon source is selected from one or more of glucose, sucrose, fructose, xylose, arabinose and mannose.

[0047] In particular, the carbon source in the growth medium is selected from raw materials, for example from the group consisting of unprocessed glycerol, starchy plant raw material hydrolysates or lignocellulosic plant raw material hydrolysates.

[0048] The specific method described in the present invention includes:

[0049] a) inoculating lactobacillus into a fermentation medium;

[0050] b) cultivating the lactobacillus in a growth medium to accumulate biomass;

[0051] c) cultivating the lactobacillus in a production medium to produce a cellular metabolite or chemical substance; and

[0052] d) Isolation and purification of cellular metabolites or chemicals.

[0053] In particular embodiments, the fermentation medium may be a growth medium, and / or culturing steps b) and c) may be employed in a single batch or in separate batches.

[0054] According to one specific aspect, a method for producing 1,3-propylene glycol from unprocessed glycerol comprises,

[0055] a) inoculating lactobacillus into a fermentation medium with unprocessed glycerol as a carbon source,

[0056] b) cultivating the lactobacillus in a growth medium to accumulate biomass,

[0057] c) cultivating the lactobacillus in a production medium to produce 1,3-propanediol, and

[0058] d) Isolation and purification of 1,3-propanediol.

[0059] According to a specific aspect, lactic acid is produced from glucose and / or xylose derived from lignocellulose or lignocellulose hydrolysate, comprising

[0060] a) inoculating lactobacillus into a fermentation medium with lignocellulose or lignocellulose hydrolysate as a carbon source,

[0061] b) cultivating the lactobacillus in a growth medium to accumulate biomass,

[0062] c) cultivating the lactobacillus in a production medium to produce lactic acid, and

[0063] d) Isolation and purification of lactic acid.

[0064] According to a specific aspect, mannitol is produced from fructose (e.g., extract or hydrolyzate) of sugar beet, including

[0065] a) inoculating lactobacillus into a fermentation medium with beet extract or hydrolyzate as a carbon source,

[0066] b) cultivating the lactobacillus in a growth medium to accumulate biomass,

[0067] c) cultivating the lactobacillus in a production medium to produce mannitol, and

[0068] d) Isolation and purification of mannitol.

[0069] In particular, the methods described herein provide for inoculating the bacteria directly into a growth medium. Fermentation medium can be used as the growth medium.

[0070] In one specific embodiment, lactobacilli grown on raw materials (e.g., unprocessed glycerol, lignocellulose, or lignocellulose hydrolysate) are cultured in batches to accumulate biomass, and subsequently fed with a purified or unpurified carbon source (e.g., containing carbohydrates such as glycerol), optionally with or without glucose and / or xylose, at a specific feed rate, to accumulate cellular metabolites or chemicals, such as 1,3-propanediol or lactic acid.

[0071] Specifically, a lactobacillus strain is a natural strain that can be isolated from a natural source 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 in nature and are therefore artificial or man-made.

[0072] According to a specific aspect, the Lactobacillus cell is an isolated naturally occurring Lactobacillus bacterium, strain, progeny or derivative thereof.

[0073] In particular, the Lactobacillus cell is from any of the Lactobacillus strains deposited as DSM 33056, DSM 33057, DSM 33058, DSM 33059 or DSM 33060, or a progeny or derivative of any of the foregoing.

[0074] Specific embodiments relate to lactobacilli derived from selected strains, including strain DSM 14421 (Leibniz Institut DSMZ-German Collection of Microorganisms, Brunschweig, Germany; Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig) Genbank accession number: AF264701 (16S rRNA), e.g., AF264701.2), strains LMG 19667, LMG 19668 (LMG from BCCM / LMG Laboratory of Microbiology, Universiteit Gent (UGent), The Netherlands), strain G77 (CUPV: Collection of the University of the Basque Country (Collección de la Universidad del Pais Vasco) (Spain), Journal of Food Protection (2006) 69, 161-169), Electronic Journal of Biotechnology The same strains as KKP 2057p (Collection of Industrial Microorganisms, Waclaw Dabrowski Institute of Agricultural and Food Biotechnology, Volume 26, March 2017, 60-63) and strain 1Z described in Benef Microbes. 2014 Dec; 5(4): 471-81 are used, for example, natural cell lines or recombinant production cell lines are used.

[0075] According to a specific embodiment, genetically engineered or recombinant Lactobacillus strains are used. A specific embodiment refers to strains that have been mutagenized and selected for improved bioconversion.

[0076] In particular, lactobacilli are used as genetically engineered or recombinant strains, for example, which are modified to produce metabolites including chemical substances, preferably to process raw materials or waste materials, for example to produce isolated fermentation products.

[0077] In particular, genetically engineered or recombinant Lactobacillus strains can be used, preferably host cells modified to overexpress homologous sequences and / or transformed with vectors containing heterologous sequences, such as 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, titer, or productivity of the biotransformation product, e.g., by overexpressing one or more genes that form the target cellular metabolite.

[0078] More specifically, the host cell is engineered to increase the yield of 1,3-propanediol from the bioconversion of unprocessed glycerol and / or the productivity of the host cell, such as the specific productivity and / or volumetric productivity.

[0079] For example, the host cell is engineered to overexpress NADPH-dependent 1,3-propanediol oxidoreductase.

[0080] The present invention also provides a Lactobacillus (L. diolivorans) strain which is any of the Lactobacillus strains deposited as DSM 33056, DSM 33057, DSM 33058, DSM 33059 or DSM 33060, or a progeny or derivative of any of the foregoing.

[0081] In particular, derivatives of these strains contain one or more mutations for overexpression of homologous sequences and / or transformation of bacteria with vectors containing heterologous sequences to increase production of cellular metabolites.

[0082] These strains, progeny or derivatives are particularly useful in the methods described herein.

[0083] According to one specific aspect, the present invention provides a method for bioconverting a carbon source into cellular metabolites using Lactobacillus cells in cell culture via an aerobic fed-batch fermentation process.

[0084] 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).

[0085] In particular, the lactobacillus cells described herein are used to bioconvert glycerol to 1,3-propanediol, preferably, wherein the glycerol is biodiesel-derived unprocessed glycerol or glycerol of less than technical grade purity 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).

[0086] According to a specific aspect, lactobacilli are used to process raw materials or waste materials to produce isolated fermentation products, such as cellular metabolites or chemicals, such as metabolites produced by the organism's (native, wild-type or recombinant) metabolic pathways.

[0087] According to a specific aspect, cellular metabolites are chemical substances produced by biotransformation of a carbon source in a bacterial cell culture and purified from the cell culture or cell culture fraction.

[0088] Such chemicals can be final products, industrial grade chemicals or chemicals of higher purity, such as USP grade chemicals, or intermediates for producing derivatives (including chemical substances, solvents or polymers). Preferred chemicals are produced as purified chemicals by preparative separation and / or purification, more preferably on a large scale or industrial scale. Specifically, chemicals can be produced in large quantities and are referred to as bulk materials or bulk chemicals. Chemicals can also be produced as specialty chemicals for pharmaceutical ingredients, bactericides and technical applications.

[0089] Cellular metabolites can be obtained at high concentrations. For example, the 1,3-propanediol concentration during or at the end of the production phase is preferably at least 40 g / L, more preferably at least 60 g / L, more preferably at least 80 g / L, and more preferably at least 100 g / L.

[0090] The methods described herein provide for high yield production. Specifically, chemicals, such as 1,3-propanediol, are produced in high yields of at least 40% or 50%, as determined below.

[0091] In particular, the product yield can 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, and the product yield is preferably at least 40%, more preferably at least any one of 50%, 60%, 70%, 80%, 85%, 90% or 95%.

[0092] The product yield can be calculated based on the 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, and the product yield is preferably at least 40%, more preferably at least any of 50%, 60%, 70%, 80%, 85%, 90% or 95%.

[0093] Preferably, the cellular metabolites are produced as a high purity material, specifically, a purity of at least 95%, preferably a purity of at least 96%, preferably a purity of at least 97%, preferably a purity of at least 98%, even more preferably a purity of 99%, and most preferably a purity of 99.5% (w / w).

[0094] The preferred purity grade is at least technical grade (generally considered to be greater than 97% (w / w) purity). Particularly preferred is polymer grade (generally considered to be greater than 98% (w / w) purity).

[0095] In particular, a priority is the large-scale production and isolation of cellular metabolites, for example on an industrial scale. Thus, as used herein, the term "isolation" specifically refers to the preparative isolation and purification of cellular metabolites from a fermentation system in sufficient quantities for further use. Thus, such preparative isolation is in contrast to analytical determination of fermentation products, which is performed solely for analytical purposes.

[0096] The preferred cell lines exhibited maximum volumetric productivity and specific productivity of 0.8 g / L / h and 0.15 g / g cell mass / h, respectively, for the production of cellular metabolites or chemicals (e.g., 1,3-propanediol). The preferred cell lines exhibited average volumetric productivity and specific productivity of 0.5 g / L / h and 0.1 g / g cell mass / h, respectively, for the production of cellular metabolites or chemicals (e.g., 1,3-propanediol).

[0097] According to a specific embodiment, the present invention relates to the use of lactobacilli in a range of bioconversion processes, for example as platform microorganisms for converting carbohydrates from raw materials comprising at least two different carbohydrate sources (e.g., comprising different types of low-purity carbohydrates) into cellular metabolites or chemicals. Evidence indicates that the lactobacilli can tolerate growth on different raw materials and can utilize a variety of carbon sources for growth and / or production of cellular metabolites or chemicals. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1 Agarose gel preparation of genomic DNA of three lactobacilli strains: DSM 14421; LMG 19668 and Vogelbusch 1050 (deposited under DSM 33056). DETAILED DESCRIPTION

[0099] Specific terms used throughout the specification have the following meanings.

[0100] The term "aerobic reactor system" as used herein refers specifically to a production unit comprising one or more reactors, including a production reactor containing a production cell culture (which can be, for example, a flow-through reactor used in a fed batch process or a continuous process, or a closed container used in a batch process), i.e., a cell culture during the production phase. Such a production reactor can be an airtight container. The airtight container can have one or more vents to discharge waste gases to be released and to ensure that the pressure accumulated in the container is minimized. The aerobic reactor system is specifically equipped with one or more devices for introducing feed culture medium into the production reactor to feed the cell culture during the production phase; and separating or collecting the cell culture medium or supernatant containing the target cell metabolites.

[0101] Specific reactor systems provide for commercial-scale cell culture. In some embodiments, the culture is at least about 100 L, or at least about 200 L, or at least about 500 L, or at least about 1,000 L, or at least about 10,000 L, or at least about 100,000 L, or at least about 500,000 L. In some embodiments, the culture is about 300 L to about 1,000,000 L.

[0102] According to a low-cost culture strategy, the cell lines are cultured under aerobic culture conditions and optionally employ an aerobic fed-batch fermentation protocol. For this reason, aerobic reactor systems typically do not include components or devices to protect the cell culture from oxygen, such as by sparging anaerobic gas.

[0103] When cultivating lactobacillus cell culture in aerobic reactor system, cell culture may still only include a small amount of detectable dissolved oxygen measured in production phase cell culture medium, or the dissolved oxygen below the detection limit. This is because lactobacillus may have the ability to consume oxygen contained in cell culture medium or feed in the production phase. Hypoxia level generally means that the level of oxygen is lower than the level in the oxygen-saturated liquid phase of cell culture. Hypoxia level can be any one of saturated dissolved oxygen concentration (oxygen saturation is 100%) in the liquid phase at about 0.1% to about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%, or below the detection limit. In a specific embodiment, cell culture medium comprises, for example, about 4ppb to about 8ppb of dissolved oxygen in the culture medium.

[0104] Method described herein specifically refers to the cell culture under aerobic culture condition, the special difference between itself and anaerobic fermentation is that it uses the aerobic reactor system that does not comprise ventilated production reactor or fermentation vessel, and it will introduce inert gas or anaerobic gas and be used for purging, or removes air (or oxygen) from container.Anaerobic fermentation generally comprises the purification step that anaerobic gas (for example being selected from the gas of helium, nitrogen, inert gas and combination thereof) is introduced into the production chamber to finish.This will especially avoid in aerobic reactor system.Compared with anaerobic fermentation, by avoiding using this ventilated production reactor or fermentation vessel, production cost will significantly reduce.

[0105] According to existing methods, Lactobacillus is bioconverted using an anaerobic reactor system under oxygen-protected conditions, using nitrogen for ventilation and purging, to produce cellular metabolites. This is essential because the key enzyme glycerol dehydratase, which is present in the anaerobic culture of Lactobacillus, is highly sensitive to oxygen, necessitating growth conditions that favor anaerobic bacterial proliferation, particularly during the production phase.

[0106] Shown that lactobacillus can be cultivated in aerobic reactor system, and need not use inert or anaerobic gas ventilation, therefore, lactobacillus can be cultivated under aerobic and keeping hypoxic condition. Surprisingly, when cultivating in aerobic reactor system according to method described herein, lactobacillus can withstand this condition. Although carried out ventilation for anti-oxygen protection, bioconversion is still very effective, and has produced target cell metabolites with high yield. Thereby, a kind of low-cost production method can be provided, and expensive ventilation equipment and ventilation process can be avoided.

[0107] Conveniently, certain lactobacillus strains are used which are novel isolates and deposited as further described herein, in particular the L. diolivorans strains deposited as DSM 33056, DSM 33057, DSM 33058, DSM 33059 or DSM 33060.

[0108] The deposited strains have been shown to be easy to handle and surprisingly insensitive to oxygen, meaning they can be handled in a fully aerated environment without losing their growth or productivity. This oxygen insensitivity is even more surprising in cell culture, offering significant advantages when producing cellular metabolites during the production phase. The deposited strains grow well under the described conditions and readily convert pharmaceutical-grade or sub-industrial-grade glycerol into 1,3-propanediol.

[0109] The aerobic reactor system used herein can be in the form of a static fermentation or a continuous fermentation system, which provides a favorable environment for the methods described herein to maintain optimal bioconversion and synthesis of the target metabolites. In certain cases, the use of inert or anaerobic gases to purge the cell culture or to clear the reactor is avoided.

[0110] Typically, the culture medium used in such cell cultures, such as growth medium or feed medium, can be in the form of an aqueous solution or slurry and introduced into the reactor without providing an anaerobic environment.

[0111] As used herein, " reactor " can comprise fermentor tank or fermentation unit, or other any reaction vessel, and term " reactor " can be used interchangeably with " fermentor tank ".For example, production reactor or bioreactor unit can carry out following one or more, or all: feed nutrient and / or carbon source, the inlet and outlet flow of fermentation or cell culture medium, separate gas phase and liquid phase, maintain reaction temperature, maintain oxygen and CO2 level, maintain pH level, agitate (for example, stir), and / or clean / sterilize.The exemplary production reactor that uses in aerobic reactor system is not injected suitable inert gas usually.For example, reactor system can comprise multiple reactors in system, may comprise multiple reactor systems in facility simultaneously.In various embodiments, reactor is applicable to batch, semi-batch feeding, batch feeding, perfusion and / or continuous fermentation process.Suitable reactor can be multipurpose, single use, disposable or non-disposable, and can be prepared by any suitable material (comprising metal alloy (for example stainless steel and Inconel), plastics and / or glass).

[0112] In embodiments, devices, facilities and methods described herein can also include any suitable unit operations and / or other unmentioned equipment, such as operations and / or equipment for separating, purifying and separating biosynthetic products. Any suitable facilities and environments can be used, such as traditional stick-built facilities, modular, removable and temporary facilities or any other suitable structures, facilities and / or layouts. In addition, unless otherwise indicated, devices, systems and methods described herein can be placed and / or performed in a single location or facility, or can be selected to be placed and / or performed in a single or multiple locations and / or facilities.

[0113] Suitable culture techniques can include culturing in a bioreactor starting with a batch phase, followed by a short exponential fed-batch phase at a high specific growth rate, followed by a fed-batch phase at a low specific growth rate. Another suitable culture technique can include a batch phase, followed by a fed-batch phase at any suitable specific growth rate or combination of specific growth rates, for example, changing from a high growth rate to a low growth rate during the production phase. Another suitable culture technique can include a batch phase, followed by a continuous culture phase with a low dilution rate.

[0114] As used herein, the term "biotransformation" is understood to be the cellular biosynthetic process of a compound, such as an organic carbon source or carbohydrate, into a cellular metabolite. A compound can be biotransformed by cells in cell culture. The production of such a compound by cell culture is also referred to as "in vivo production" or "in vivo biotransformation." It is understood that such biotransformation in cell culture is "ex vivo," meaning that it does not involve higher organisms, animals, or humans. As further described herein, specific biotransformation processes are carried out in bacterial cell culture to produce cellular metabolites, such as chemicals, particularly chemical substances as further described herein.

[0115] As used herein, the term "carbon source" refers to a carbon substrate, particularly a fermentable carbohydrate, such as a carbohydrate used in a bioconversion process, and specifically includes a source carbohydrate that can be metabolized by a host organism or production cell line. As used herein, the term "carbohydrate" is broad, for example, including sugars, sugar acids, and their derivatives, for example, carbohydrates that conform to the general formula (CHO) n Polyhydroxy aldehydes or ketones and their derivatives. Specifically include aldehydes (such as glyceraldehyde), ketones (such as dihydroxyacetone) and hydrogenated derivatives (such as glycerol). Specifically include polyols, preferably at least three-carbon polyols, such as sorbitol, mannitol or glycerol. The specific carbohydrate used as a carbon source is supplied in purified form or as raw materials and is selected from monosaccharides, oligosaccharides, polysaccharides, polyols and their derivatives.

[0116] The carbon source used for bioconversion as described herein can be a purified carbon source or a raw material (e.g., a carbon source comprising carbohydrates in a mixture with impurities), and can also be understood as a "fermentable carbon source" or "fermentable carbohydrate" because it is bioconverted to cellular metabolites by fermentation.

[0117] As used herein, the term "cell line" refers to an established clone of a specific cell type that has acquired the ability to proliferate over a long period of time. The term "host cell line" refers to a cell line used to express products of endogenous or recombinant genes or 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 to be cultured in a bioreactor to obtain a production process product. Suitable production cell lines can produce some useful intermediates and products by saccharifying low molecular weight sugars produced by fermentation-treated biomass materials. Suitable production cell lines can also produce the necessary enzymes to saccharify the treated biomass materials while producing useful intermediates and products. For example, fermentation or other biological processes can produce alcohols, organic acids, hydrocarbons, hydrogen, proteins, or mixtures of any of these substances.

[0118] As used herein, the term "cell culture" or "culturing" or "cultivating" in relation to Lactobacillus cells refers to maintaining the bacterial cells in an artificial environment (e.g., an in vitro environment) under conditions that are conducive to cell growth, differentiation or continued survival, in an active or dormant state, in particular in a controlled bioreactor, according to methods known in the art.

[0119] When using a suitable culture medium to cultivate cell cultures, the cells are contacted with a culture medium or substrate in a culture vessel under conditions suitable for supporting the cultivation of the cells in the cell culture. As described herein, culture medium that can be used for cell growth is provided. Standard cell culture techniques are well known in the art.

[0120] The cell cultures described herein particularly utilize techniques for producing target metabolites by bioconversion, such as obtaining a product in the cell culture medium that can be separated from the cell biomass (referred to herein as a "cell culture supernatant") and, optionally, can be isolated and purified to obtain a higher purity product.

[0121] Cell culture media provides the nutrients necessary to maintain cell survival and growth in a controlled, artificially mediated, in vitro environment. The characteristics and composition of cell culture media can be varied according to the specific cell needs. Important parameters include osmotic pressure, pH, and nutrient formulation. Nutrient addition can be accomplished continuously or discontinuously according to methods known in the art.

[0122] Batch culture is a cell culture mode in which all nutrients required for the cultured cells are contained in the initial culture medium, eliminating the need for additional nutrients during the fermentation process. In contrast, in a fed-batch process, one or more nutrients are added to the culture via feed after the batch phase. While the feed mode is crucial in most processes, the cell culture and methods described herein are not limited to a single mode of cell culture.

[0123] In certain embodiments, the cell culture process utilizes a fed-batch process.

[0124] In another embodiment, the host cells described herein are cultured in a continuous mode. Continuous fermentation processes are characterized by the addition of fresh culture medium to the bioreactor at a defined, constant, and continuous rate, while the culture fluid is removed from the bioreactor at the same defined, constant, and continuous rate. By maintaining the culture medium, feed rate, and removal rate at the same constant level, the cell culture parameters and conditions in the bioreactor are maintained constant.

[0125] As described herein, a stable cell culture is one that maintains the genetic properties of the cells, in particular, maintains a high biosynthesis rate and production level, for example 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. In particular, the present invention provides a stable Lactobacillus cell culture that has great advantages when used to produce target cell metabolites on an industrial scale.

[0126] As used herein, the term "cellular metabolite" or "target cellular metabolite" refers to a metabolite produced by a cell through the bioconversion of a carbon source, eg, through a metabolic pathway to produce a primary or secondary metabolite.

[0127] The biotransformation products produced by the methods described herein are specifically 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 lead to immediate death of the organism, but rather to long-term impairment of the organism's viability, reproductive capacity, or aesthetics, or may not significantly change at all. Specific secondary metabolites are typically limited to a narrow set of species within a phylogenetic group.

[0128] As used herein, the term "chemical" refers to chemical substances or compounds, in particular low molecular weight organic molecules (called "small organic molecules"), such as those used in the chemical industry, pharmaceutical industry, agriculture, cosmetics industry, food industry and feed industry.

[0129] Compounds specifically produced according to the methods described herein can include chemicals in small or large quantities, of low or high purity, including bulk chemicals, fine chemicals, and specialty chemicals. The term encompasses final products as well as intermediates for producing derivatives, such as reaction products, including monomers for polymer synthesis.

[0130] As used herein, the term "crude glycerol", also known as "unprocessed glycerol", is a by-product from the production of fatty acids, bioethanol or biodiesel or from soap production processes, also known as industrial glycerol derived from (or originating from) biodiesel production. Crude glycerol can be used as a raw material or source material and can also be used as a carbon substrate in some biotechnology fields. It contains glycerol (usually in the range of 40-88%, such as about 50% (w / w), in some cases up to 95%) as well as salts, soaps and other impurities, including water and methanol. The typical composition of crude glycerol, for example, is obtained from NOVAOL Srl (Milan, Italy), from The unprocessed glycerol in the product is provided as crude glycerol obtained from a biodiesel production process, having the following composition: 85-95% (w / w) glycerol, 5-10% (w / w) organic / inorganic acids, and 0-1.5% (w / w) methanol. Another example of crude glycerol is thin stillage (raw material produced by ethanol distillation or a by-product obtained from bioethanol distillation) or thick stillage (concentrated thin stillage, such as that obtained by evaporating thin stillage into thick stillage).

[0131] In particular, the unprocessed glycerol used in the methods described herein is obtained from waste materials of biodiesel, bioethanol or fatty acid production, for example, from different seed oil feedstocks including palm oil, jatropha, mustard, rapeseed, canola, crambe, soybean and waste cooking oil.

[0132] Specifically, crude glycerol is recovered from the product of fatty acids, biodiesel, bioethanol or soap without further processing, for example, by separation from fatty acids, biodiesel and soap, respectively. Nevertheless, the term "crude glycerol" also includes crude glycerol that has been fractionated or processed, for example, including by filtration, ion exchange, chemical addition and / or fractionated vacuum distillation to obtain materials of different commercial grades. According to a specific embodiment, the crude glycerol can be treated to neutralize the material, for example, to obtain a pH range between 6 and 8, preferably about 7. Thereby, an organic phase containing fatty acids and an aqueous phase containing glycerol in addition to impurities are obtained. Although this fractionated crude glycerol still contains glycerol with a purity lower than that of technical grade glycerol, specifically contains unwanted ash, it can still be used as a carbon source in the method described herein.

[0133] According to a certain embodiment, the crude glycerol is pasteurized or sterilized, for example, by autoclaving the crude glycerol at 121° C. for 10 to 100 minutes, for example, about 20 minutes, before using it as a carbon source for bioconversion. According to another embodiment, the crude glycerol is pasteurized by heating the material to at least 80° C. for 10 to 100 minutes, for example, about 15 minutes (“about” means + / - 1 or 2° C.), before using it as a carbon source for bioconversion.

[0134] The purity of technical grade glycerol is usually greater than 97% (w / w).

[0135] In particular, the crude glycerol used as a carbon source for bioconversion is characterized in that the glycerol purity is lower than that of technical grade glycerol, for example, the purity is lower than 97%, or lower than 96%, specifically lower than 95%, more specifically lower than 94%, more specifically lower than 93%, more specifically lower than 92%, more specifically lower than 91%, more specifically lower than 90% (w / w).

[0136] In particular, crude glycerol use is characterized by the content of impurities, such as ash, as determined by standard assays.

[0137] The ash content in crude glycerol mainly comes from the sodium in the catalyst used in the biodiesel production process, and depending on the oil source and the biodiesel production process, the ash content in crude glycerol is generally between 0.25% and 5.50%, and in some cases as high as 8% (Thompson et al. Applied Engineering in Agriculture 2006, Vol. 22(2): 261-265).

[0138] As used herein, the term "derivative" in relation to a Lactobacillus strain refers to a strain obtained from a parent strain by, for example, mutagenesis and selection or directed mutagenesis.

[0139] Specifically, derivatives of the Lactobacillus strains involved in this article can be produced by limited mutagenesis, for example, maintaining a certain sequence similarity throughout the genome, such as at least any of 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0140] It is understood that the biotransformation products produced by the methods described herein are cellular metabolites or chemical substances, or derivatives of any of the foregoing, which are conveniently produced upon isolation and, optionally, purification. "Derivatives" of cellular metabolites or chemical substances are specifically understood to be compounds derived from the products of the biotransformation process, for example, by chemical, physical or biological synthesis, including polymerization, acetylation, methylation or phosphorylation.

[0141] As used herein, the term "Lactobacillus diolivorans" or "L. diolivorans" refers to a species of the genus Lactobacillus, either an isolated strain, a wild-type bacterium, or a derivative thereof (e.g., a genetically engineered producer cell line) first described by Krooneman et al. (International Journal of Systematic and Evolutionary Microbiology 2002, 52, 639-646).

[0142] The "deposited strains" of Lactobacillus referred to herein are as follows.

[0143] The following Lactobacillus (L. diolivorans) strains were isolated in Austria and deposited on February 26, 2019, by Vogelbusch GmbH, Vienna, Austria, at the DSMZ - German Collection of Microorganisms, Mascheroder Weg 1b / Inhoffenstraße 7B, 38124 Brunswick (DE).

[0144] The deposit relates to lactobacillus strains and cultures, which are characterized as follows.

[0145] DSM 33056 (also known as Lactobacillus diolivorans Vogelbusch 1050) is characterized by its ease of handling and oxygen insensitivity, meaning it can be handled in a fully aerated environment without loss of growth or productivity. In particular, the strain exhibits outstanding 1,3-propanediol production in mixtures of glycerol with one or more of glucose, fructose, or mannose.

[0146] DSM 33057 (also known as Lactobacillus HH) is characterized by ease of handling and oxygen insensitivity, meaning it can be handled in a fully aerated environment without loss of growth or productivity. It exhibits outstanding 1,3-propanediol production, particularly in mixtures of glycerol with either glucose or fructose. In particular, it is one of the best performing strains for 1,3-propanediol production in mixtures of glycerol and sucrose.

[0147] DSM 33058 (also known as Lactobacillus HM) is characterized by its ease of handling and oxygen insensitivity, meaning it can be handled in a fully aerated environment without loss of growth or productivity. In particular, the strain exhibits outstanding 1,3-propanediol production in mixtures of glycerol with one or more of glucose, fructose, or mannose.

[0148] DSM 33059 (also known as Lactobacillus HS) is characterized by its ease of handling and oxygen insensitivity, meaning it can be handled in a fully aerated environment without loss of growth or productivity. It exhibits outstanding 1,3-propanediol production performance, particularly in mixtures of glycerol with either glucose or fructose. The strain's performance in 1,3-propanediol production is particularly optimal in mixtures of glycerol with xylose. It also performs best in mixtures of glycerol and mannose.

[0149] DSM 33060 (also known as Lactobacillus HF) is characterized by its ease of handling and oxygen insensitivity, meaning it can be handled in a fully aerated environment without loss of growth or productivity. In particular, the strain exhibits outstanding 1,3-propanediol production in mixtures of glycerol with one or more of glucose, fructose, or mannose.

[0150] As used herein, the term "final product" refers to a cellular metabolite or a quality-controlled chemical substance that is produced and optionally processed into a product for further manufacturing or industrial purposes or as a consumer product.

[0151] 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 that has been engineered to improve a production process or to enable the production of a new product. The term is in contrast to a "wild-type" organism, which is typically not genetically engineered. The term "wild-type" as used in this disclosure typically refers to "isolated from a natural source," such as Lactobacillus diolivorans DSM 14424 or DSM 14421, or Lactobacillus diolivorans LMG 19668, or Lactobacillus diolivorans G77, or any of the deposited strains DSM 33056, DSM 33057, DSM 33058, DSM 33059, or DSM 33060, including any progeny thereof.

[0152] As used herein, the term "intermediate" refers to a chemical substance formed as a product of a bioconversion process that can be isolated from the fermentation broth and further processed to form a derivative or final product.

[0153] As used herein, the term "isolated" in relation to a cellular metabolite, a chemical substance, or a biosynthetic or biotransformation process refers to a substance that is separated or purified (especially by a preparative method) from at least one impurity to obtain a product that is completely separated from the environment with which it was associated prior to isolation, thereby being in a "substantially pure" form. "Isolated" does not necessarily mean excluding the presence of artificial or synthetic mixtures with other compounds or materials, or impurities that do not interfere with the essential activity, such as may be present due to incomplete purification.

[0154] As used herein, the term "isolate" refers to a bacterial strain.

[0155] As used herein, the term "metabolic pathway" refers to a biochemical reaction that occurs within an organism, for example, the metabolism of a particular compound or a microbial enzyme pathway that produces cellular metabolites, particularly small organic molecules. A metabolic pathway refers to all biosynthetic, modification, and degradation pathways of a compound within a cell. Metabolic engineering refers to the introduction, deletion, and modification of a microbial metabolic pathway, specifically through the use of appropriate recombinant techniques, and is widely used to efficiently produce desired metabolites and biomolecules. Even novel synthetic products can be obtained through metabolic engineering of host cells.

[0156] As used herein, the term "mutagenesis" refers to a recombinant construct or organism with a mutated nucleic acid, thereby obtaining a variant thereof with at least one change in a non-coding region or a coding region. Mutagenesis can be by random, semi-random or site-directed mutagenesis.

[0157] As used herein, the term "overexpression" used in relation to host cells, particularly recombinant host cells, 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 overexpression of homologous (endogenous) as well as heterologous sequences or proteins, and is particularly intended to increase the productivity of the cell to produce a bioconversion product, e.g., by 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.

[0158] As used herein, term " production phase " refers to a certain stage that cell culture produces biotransformation product. The production phase can specifically be after the growth phase, and the growth phase cell culture accumulates biomass by growing cells. The cultivation of growth and production phase can be carried out conveniently by batches, fed-batch and continuous culture process. The carbon source that is suitable for being used as the substrate of biological conversion to cellular metabolites can be included in the feed of batch feed process, for example, under the condition that carbon substrate is limited, namely under the condition that growth is restricted and in production mode, use a limited amount of carbon source to maintain production cell line. This limited amount can be used for batch feed process, wherein carbon source is included in feed medium, and is supplied to culture with low feed rate, for continuous energy delivery, for example, keeping biomass in low specific growth rate and producing biosynthesis or fermentation product. In the production phase of cell culture, usually fed-batch medium is added in fermented liquid.

[0159] As used herein, the term "raw material" specifically refers to any complex carbon source or carbohydrate material, in particular carbohydrate-rich biomass, including low-purity carbohydrates in monomeric or polymeric form, such as from bioenergy crops, industrial crops, agricultural residues, municipal waste, industrial waste, yard waste, wood, straw, chitosan containing residues from shellfish. Specific examples of raw materials are unprocessed (crude) glycerol, sugar cane, sugar beets, starchy plants, cellulose, hemicellulose, lignocellulose, lignocellulose hydrolysates or chitosan.

[0160] The complex carbohydrate mixture in the raw material generally comprises at least two different organic carbon sources in large amounts, for example, each carbon source in an amount of at least 2%, particularly at least 3%, more particularly at least 4%, more particularly at least 5% (w / w).

[0161] In particular, the raw materials used in the methods described herein are characterized by a fermentable carbohydrate content having a purity lower than technical grade (industrial grade purity > 97% (w / w)), for example lower than 96%, specifically lower than 95%, more specifically lower than 94%, more specifically lower than 93%, more specifically lower than 92%, more specifically lower than 91%, more specifically lower than 90% purity (w / w).

[0162] Specifically, the raw materials used in the methods described herein are characterized by their impurities, such as ash content, as determined by standard methods. Exemplary standard methods determine the residue left after the combustion of organic matter. Ash content is generally considered an approximate measure of the mineral content and other inorganic matter content of biomass.

[0163] A typical standard method for determining the ash content in raw materials (e.g., crude glycerol) is the gravimetric method, as described, for example, in the International Union of Pure and Applied Chemistry, Applied Chemistry Division, Commission on Oils, Fat and Derivatives, "Standard Methods for the Analysis of Oils, Fat and Derivatives," 6th edition, 1st Supplement: Part 2 (1980), Section III, Glycerol, prepared and published by A. HAUTFENNE, Catholic University of Louvain, Louvain-la-Neuve, Belgium (6). th Edition,1 st Supplement: Part 2 (1980), SECTION III. GLYCERINES, Prepared for publication by A. HAUTFENNE, Université Catholique de Louvain, Louvain-la-Neuve, Belgium).

[0164] According to this method, the test consists of burning the test part, igniting the organic matter and weighing the residue. The ash content is defined as the amount of ash expressed as mass percentage (n / m).

[0165] The raw materials used in the bioconversion methods described herein are 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).

[0166] More specifically, the raw materials used include fermentable carbohydrates of less than technical grade purity and with a certain level of impurities (eg, ash, as further described herein).

[0167] Specifically, the raw material used is crude glycerol, which is characterized by a glycerol purity lower than that of technical-grade glycerol and contains a certain amount of impurities (such as ash, as further described herein).

[0168] The raw material that is used for carbon source bioconversion into cellular metabolites as described herein is the crude material that does not need to be further processed that is recovered from various sources. Nevertheless, term " raw material " also comprises the crude material through fractionation or processing, for example comprises by filtering, centrifuging, ion exchange, chemical interpolation, enzyme treatment and distillation (for example fractional vacuum distillation) to obtain the material of different commercial grades. Preferably, before the raw material is used, adopt method as described herein to process and handle. Preferred processing comprises pasteurization, sterilization (for example passing through autoclaving), handles (for example obtains 6 to 8 pH, preferably about 7) with chemicals (for example acid or alkali), or fractionation (for example reduces the content of unwanted organic acid or alcohol).

[0169] Such fractionated or processed crude material also contains fermentable carbohydrates of less than technical grade purity and undesirable ash and, therefore, can be used as a raw material in the methods described herein.

[0170] According to a certain embodiment, the raw material is pasteurized or sterilized, for example, by heat treatment at 80°C for 10 to 100 minutes (e.g., about 15 minutes ("about" means + / - 1 or 2°C)), or by autoclaving the material at 121°C for 10 to 100 minutes (e.g., about 20 minutes ("about" means + / - 1 or 2°C)).

[0171] In particular, the methods described herein include the additional step of sterilizing the raw materials before adding them to the cell culture.

[0172] In the present invention, the term "sequence identity" means that two or more nucleotide sequences have identical or conserved base pairs (to a certain extent, up to 100%) at corresponding positions.

[0173] "Percent (%) identity" of a gene or genomic nucleotide sequence is defined as the percentage of nucleotides that are identical to the nucleotides in the candidate DNA sequence and the DNA sequence to which the candidate DNA sequence is compared, after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent nucleotide sequence identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0174] As used herein, the term "recombinant" means "produced by or as a result of genetic engineering." Thus, a "recombinant microorganism" comprises at least one "recombinant nucleic acid." A recombinant microorganism may be a mutant produced by mutagenesis using an appropriate method and / or specifically comprise an expression vector or cloning vector, or it may have been genetically engineered to comprise a recombinant nucleic acid sequence or a recombinant metabolic pathway to produce a cellular metabolite at high yield or to produce a novel cellular metabolite that is not produced in large quantities by wild-type cells (before recombination).

[0175] As described herein, lactobacillus unexpectedly has tolerance to aerobic culture conditions, so even without nitrogen or other protective gases, lactobacillus strains also can produce cellular metabolites with high yield by biotransformation. Various carbon sources can be used as substrates for biotransformation, even those unprocessed materials or waste materials and low-purity materials. Therefore, the bacterial classification can be used as a production cell line to produce the product of the biotransformation process in an economical and efficient manner. The specific lactobacillus strains preferably used are provided. Specifically, lactobacillus strains are easy to metabolize a series of substrate sugars or polyols or other carbohydrates to convert (biotransformation) into cellular metabolites or chemical substances. Particularly preferred carbohydrates are glycerol, especially crude glycerol.

[0176] A particularly preferred method comprises the following steps:

[0177] (i) pretreatment of raw materials for purification and / or sterilization,

[0178] (ii) fermentation of raw materials for cell growth and cell metabolism or accumulation of chemical substances,

[0179] (iii) separation of cellular metabolites or chemicals, and

[0180] (iv) Purification of cellular metabolites or chemicals.

[0181] Specifically, the production process for producing 1,3-propylene glycol from unprocessed glycerol comprises the following steps:

[0182] (i) pre-treatment of the crude glycerol for purification and / or sterilization, for example, by pH adjustment and fractionation to separate the aqueous phase, and / or sterilization by autoclaving,

[0183] (ii) fermentation of raw glycerol to promote cell growth and accumulation of 1,3-propanediol,

[0184] (iii) separation of 1,3-propylene glycol, and

[0185] (iv) Purification of 1,3-propanediol.

[0186] In some embodiments, the raw material is used to grow the lactobacillus, for example, to increase the biomass in the preparation of the bioconversion process, and can also be used further in the production stage, for example, as the substrate of bioconversion or to be used for bioconversion simultaneously with another carbon source. Usually the accumulation of biomass and the production of cellular metabolites are continuous or carried out simultaneously. For growing organisms, optionally the raw material is pre-treated, for example, the lipid phase is removed, and one or more other carbon sources, for example glucose, wood sugar, arabinose, fructose, sucrose or other can be supplemented.

[0187] In certain cases, fermentation processes produce large quantities of more than one cellular metabolite, which can be isolated and purified. Thus, specific embodiments involve bioconversion of fermentable carbohydrates from a raw material, particularly in the same fermentation process, to produce purified forms of at least two different cellular metabolites.

[0188] The renewable resource to be converted includes glycerol, for example unprocessed (crude) glycerol, such as thin stillage (raw material obtained as a by-product of bioethanol distillation) or thick stillage (concentrated thin stillage, for example obtained by evaporation to thick stillage). Preferably, the unprocessed glycerol is treated before use in the fermentation process, for example, by autoclaving and separating the glycerol containing aqueous phase, to reduce the fatty acid content.

[0189] 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 starchy plant materials of various purified grades or sugar cane or sugar beet.

[0190] Cellulose glucose is another preferred carbon source. A preferred source of pentoses is hemicellulosic plant material. Specifically, the degree of purification of the hemicellulosic plant material can be relatively low.

[0191] The conversion of the carbon source is specifically carried out in a liquid phase, preferably in an aqueous environment. The conversion of the carbon source can be carried out simultaneously with the growth of the microorganism or independently of the growth of the microorganism. Therefore, cellular metabolites can accumulate in the culture medium during the growth of the microorganism or after the microorganism stops growing, or both.

[0192] The microorganisms can be inoculated into a fermentation medium, or directly into a production medium or feed, wherein the fermentation medium is a medium provided for bacterial growth, such as a growth medium.

[0193] Growth and / or production may suitably be carried out in batch mode, fed-batch mode or continuous mode.

[0194] 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 used for biomass production can, but need not be, the same as the carbon source for producing cellular metabolites.

[0195] For example, the strain can be grown on glucose or xylose to provide biomass and then fed with (unprocessed) glycerol or unprocessed glycerol and glucose, which is converted to 1,3-propanediol.

[0196] The growth medium that allows biomass accumulation generally comprises a carbon source, a nitrogen source, a sulfur source and a phosphate source. Generally, this medium also comprises trace elements, vitamins and amino acids. The medium may or may not comprise a defoamer.

[0197] Preferred nitrogen sources include ammonia; ammonium salts such as NH4Cl, (NH4)2SO4, (NH4)2CO3; nitrates such as NaNO3, KNO3; urea; and amino acids.

[0198] Preferred sulfur sources include sulfuric acid or sulfates, such as (NH4)2SO4, Na2SO4, NaHSO4, MgSO4, MnSO4, methionine, cysteine.

[0199] Preferred phosphate sources include phosphoric acid or phosphate salts, such as Na2HPO4, NaH2PO4, K2HPO4, KH2PO4.

[0200] Other typical media ingredients include yeast extract, peptone, meat extract, malt extract, corn steep liquor, corn steep liquor meal, or fish meal.

[0201] Preferably, the culture medium is supplemented with vitamin B 12 .

[0202] Typical growth media for lactobacilli include casein peptone (tryptic digest), meat extract, yeast extract, Tween 80, K2HPO4, sodium acetate (Na-acetate), (NH4)2H-citrate, MgSO4×7H2O, MnSO4×H2O.

[0203] Another typical growth medium for lactobacilli includes K2HPO4, (NH4)2H-citrate, KH2PO4, sodium chloride, ascorbic acid, potassium acetate, Tween 80, 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, niacin, calcium pantothenate, cyanocobalamin, p-aminobenzoic acid, inositol, pyridoxal HCl, riboflavin, biotin, folic acid, and FeSO4×7H2O.

[0204] A typical production medium includes a carbon source for the bioconversion, as well as sugars and optionally vitamins.

[0205] The feed to the fermentation is typically a carbohydrate solution containing up to 50 wt% carbohydrates.The feed rate can limit the inhibitory effects of the product on cell growth, so that high product yields based on the substrate supply are possible.

[0206] The fermentation is preferably carried out in a slightly acidic medium, for example, at a pH of 5.5-6.0, preferably at a pH of about 5.7 ("about" is specifically understood to mean + / - 0.1).

[0207] Typical fermentation times are about 24 to 180 hours, at temperatures ranging from 26°C to 40°C, preferably at 30°C ("about" being understood to mean + / - 1°).

[0208] The production cell lines are particularly useful on a large or industrial scale.

[0209] The specific production scale preferably uses a volume of at least 50 L, preferably at least 1 m 3 , more preferably at least 10m 3 , more preferably at least 100m 3 , most preferably at least 500m 3 .

[0210] Industrial scale production conditions refer to, for example, 100 L to 100 m 3 Batch culture in reactors of approximately 50-1000 L or larger with a typical process time of several days, or continuous production in fermenters of approximately 50-1000 L or larger with a dilution rate of approximately 0.05-0.15 h -1 .

[0211] Suitable culture techniques may include culture 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 at a low dilution rate.

[0212] Specifically, Lactobacillus strains can be used in homologous fermentation processes, wherein the cellular metabolite or end product produced (which can be a derivative of the cellular metabolite) is primarily a chemical substance, such as lactic acid or lactate. Alternatively, the process can be a heterologous fermentation, wherein the product of some other metabolic pathway is produced. Genetically engineered variants of Lactobacillus can include metabolic pathways that are not naturally present in Lactobacillus, allowing the production of a variety of cellular metabolites.

[0213] Applicable metabolic pathways that mediate the bioconversion or metabolism of carbon sources (e.g., glycerol; hexoses, such as glucose, fructose, galactose, or pentoses, such as xylose or arabinose) in lactobacilli under aerobic conditions provide for efficient conversion of carbon or carbohydrate sources to cellular metabolites. The cellular metabolites thus produced can be typical negligible products of metabolic pathways in wild-type strains. Homologous fermentation pathways can be designed by overexpressing pathways involved in the conversion of carbohydrate sources to desired cellular metabolites and / or blocking pathways that result in the synthesis of competing by-products.

[0214] For example, specific enzymes involved in overexpression are glycerol transporter, 1,3-propanediol oxidoreductase, glycerol kinase, glycerol-3-phosphate dehydrogenase, glycerol dehydrogenase, dihydroxyacetone kinase, and triosephosphate isomerase. In addition, the synthesis of unwanted byproducts (such as succinate, acetate, and ethanol) can be minimized by knocking out, deleting, or inhibiting the corresponding genes.

[0215] A specific example involves the fermentation of glucose and glycerol to 1,3-propanediol using wild-type lactobacilli in a fed-batch phase, ie, biomass accumulates during growth on glucose and fermentation is initiated when glycerol is added.

[0216] Another specific example involves the fermentation of glucose and glycerol to 1,3-propanediol using wild-type lactobacilli in a fed-batch phase, wherein biomass is accumulated while growing on glucose in the presence of glycerol, and fermentation is initiated upon feeding of glycerol and glucose.

[0217] Another specific example involves the fermentation of xylose and glycerol to 1,3-propanediol using wild-type lactobacilli, e.g., using xylose as a batch medium for growing the biomass and glycerol as a feed medium to initiate the bioconversion process.

[0218] According to another specific example, wild-type lactobacilli are cultured in batches using glucose and crude glycerol, and glucose and crude glycerol are used simultaneously in the batch culture to grow biomass and achieve bioconversion of glycerol to 1,3-propanediol.

[0219] Another example involves culturing wild-type lactobacilli on glucose in a batch phase, allowing them to grow biomass and bioconvert glucose to lactic acid.

[0220] Yet another example involves culturing wild-type lactobacilli with xylose in a batch phase, allowing them to grow biomass and bioconvert xylose to lactic acid.

[0221] We further showed that wild-type lactobacilli can be fermented with arabinose in a batch stage, allowing them to grow biomass and bioconvert arabinose into lactic acid.

[0222] According to another example, wild-type lactobacilli are fermented with fructose in a batch phase to grow biomass and bioconvert the fructose to mannitol.

[0223] According to a further embodiment,

[0224] a. 1,3-Propanediol can be produced from various mixtures of glucose and / or mannose in industrial glycerol (crude glycerol; a byproduct of fatty acid, biodiesel, bioethanol, or soap production). 1,3-Propanediol production is not limited to crude glycerol as a raw material; it can also be produced from a mixture of crude glycerol and thin stillage (from alcohol distillation) or thick stillage (thick stillage evaporated from thin stillage).

[0225] b. 3-Hydroxypropionic acid can be produced as another chemical substance using raw materials such as crude glycerol, or a mixture of crude glycerol with thin stillage or thick stillage.

[0226] c. Lactic acid can be produced as another chemical substance using any of crude glycerol, a mixture of crude glycerol and thin vinasse, thick vinasse, or lignocellulosic biomass hydrolysate (Miscantus hydrolysate).

[0227] d. Using crude glycerol as raw material, 3-hydroxypropionaldehyde can be produced as another chemical substance.

[0228] Recombinant strains of Lactobacillus LMG 19668 employing 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 an endogenous plasmid of Lactobacillus are described. The recombinant Lactobacillus LMG 19668 has been engineered to overexpress 1,3-propanediol oxidoreductase. This improves the yield of bioconversion and 1,3-propanediol biosynthesis.

[0229] According to a specific embodiment, the Lactobacillus strain is subjected to a mutagenesis and / or selection procedure, for example to obtain a strain with improved productivity, improved substrate range, improved product or substrate tolerance, improved product range or improved glycerol uptake rate.

[0230] A preferred mutagenesis method comprises 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 irradiating a culture of the lactobacillus with ultraviolet light.

[0231] A preferred selection method involves plating the bacterial culture on agar plates that exhibit selective pressure. For example, selection can involve plating the bacterial culture on agar plates containing increasing amounts of product or substrate impurities to select for strains that exhibit increased tolerance to the product or substrate impurities. Another preferred method for selecting desired bacterial strains involves sorting procedures using flow cytometry.

[0232] According to a specific aspect, the Lactobacillus (L. diolivorans) strain is genetically engineered, ie the natural genetic makeup of the organism has been altered.

[0233] This includes, for example:

[0234] ●The deletion, inactivation, or attenuation of naturally occurring genes in an organism

[0235] Overexpression of naturally occurring genes in an organism

[0236] ● Mutations in naturally occurring genes in organisms

[0237] ●Addition or expression of genes that do not occur naturally in an organism

[0238] ●Or any combination thereof.

[0239] Specific methods for genetic engineering of Lactobacillus (L. diolivorans) include:

[0240] ● Provide recombinant DNA segments, and / or

[0241] • Transforming an organism with the DNA fragment.

[0242] In general, the recombinant nucleic acids or organisms mentioned herein can be produced by recombinant techniques well known to those skilled in the art. Conventional methods of molecular biology, microbiology, and recombinant DNA technology within the skill of the art can be employed in accordance with the present invention. These techniques are explained in detail, for example, in Maniatis, Fritsch & Sambrook, Molecular Cloning: A Laboratory Manual (1982).

[0243] The example of the DNA fragmentation that is used for the genetic engineering of lactobacillus comprises plasmid, linear DNA fragment, for example PCR product, and other well known in the art.Concrete plasmid comprises the replication origin, natural promoter, selectable marker and the expression cassette of target gene of natural lactobacillus plasmid.The nucleotide sequence that can be used for engineered cell line can obtain from various sources, and the nucleotide sequence of using in the method as described herein will provide improved biotransformation process.The source of promotor is preferably from the genomic DNA of lactobacillus DSM 14421, DSM 33056, DSM 33057, DSM 33058, DSM 33059, DSM 33060, LMG 19668 or G77.

[0244] The promoter can be any suitable DNA sequence that shows transcriptional activity in the host cell and can be derived from a gene encoding a protein that is homologous or heterologous to the host cell. The promoter is preferably derived from a gene encoding a protein that is homologous to the host cell. The promoter can be an endogenous promoter or heterologous to the host cell.

[0245] Promoter sequences suitable for use in prokaryotic host cells may include, but are not limited to, promoters obtained from Lactobacillus sp., Lactococcus sp., Staphylococcus sp., Pediococcus sp., Enterobacteria sp., Streptococcus sp., and Oenococcus sp. Promoters are not limited to any particular species, provided that they can function in prokaryotic host cells, particularly in lactobacilli.

[0246] Other suitable promoter sequences for 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 the cell, for example, glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, phosphofructokinase, or enolase.

[0247] In preferred expression systems, the promoter is an inducible or constitutive promoter.

[0248] Suitable expression vectors typically contain regulatory sequences suitable for expressing the DNA encoding heterologous polypeptides or proteins 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. Regulatory sequences can be operably linked to the DNA sequence to be expressed. For example, if a promoter controls transcription of a coding sequence, then the promoter sequence is said to be operably linked to the coding sequence.

[0249] The preferred method adopts plasmid, which is pSHM. Expression vectors can include but are not limited to cloning vectors, modified cloning vectors and specifically designed plasmids. The preferred expression vector for producing recombinant lactobacilli can be any expression vector suitable for expressing recombinant genes in bacterial host cells. The recombinant expression vector can be any vector that can replicate or integrate in the genome of the host organism.

[0250] Preferably, a plasmid derived from wild-type lactobacillus is used as an expression vector.

[0251] To allow the recombinant nucleotide sequence to be expressed in the 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.

[0252] According to specific embodiment, recombinant construct is obtained by connecting the relevant gene to the vector. These genes can be stably integrated into the host cell genome, or replicated as an episomal plasmid (episomal plasmid). The vector can be transferred to the host cell by transformation. The preferred transformation method of microorganism uptake recombinant DNA fragments comprises chemical transformation, electroporation or transformation by primary plasmid. Transformant can be obtained by such carrier DNA (for example plasmid DNA), being introduced into the host, and selecting to express the relevant protein or host cell metabolite in high yield.

[0253] The polypeptide encoded by the gene can be produced using a recombinant host cell line by culturing the transformant to obtain it in an appropriate culture medium, isolating the expressed product or metabolite from the culture, and optionally purifying it by an appropriate method.

[0254] There are several preferred methods for producing one or more cellular metabolites using the strains and methods described herein. The substances can be expressed, processed, and secreted by transforming a bacterial organism with an expression vector carrying recombinant DNA encoding the 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.

[0255] Preferred host cell lines can stably maintain their genetic characteristics and maintain high production levels, for example, 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, the production level remains unchanged at least at the μg level. Stable host cells are considered a major advantage in the industrial-scale production of chemicals.

[0256] 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 the desired cellular metabolite or chemical and / or decreasing the concentration of at least one impurity.

[0257] The following separation and purification methods are preferred, in particular for preparative separations: distillation, chromatography, crystallization, filtration, centrifugation, decantation, reprecipitation or electrodialysis.Cellular metabolites or chemical substances can be obtained, for example, from fermentation broths clarified using a centrifuge.

[0258] A highly purified product can be produced which is substantially free of contaminating proteins and preferably has a purity of at least 90%, more preferably at least 95%, or even at least 98%, up to about 100%. The purified product can be obtained by purifying cell culture supernatants or from cell debris.

[0259] The isolated and purified products can be identified by conventional methods, such as HPLC, GC, MS, NMR, and infrared spectroscopy.

[0260] The foregoing description will be more fully understood with reference to the following examples. These examples, however, merely represent methods of practicing one or more embodiments of the invention and should not be construed as limiting the scope of the invention.

[0261] Example

[0262] Example 1: Preparation of MRS-medium for isolating and culturing lactobacilli:

[0263] Table 1: MRS medium

[0264]

[0265] Add 30 g / L of the desired carbon source (usually glucose) and, if necessary, 10 g / L of glycerol. For solid media, add 20 g / L of agar. Adjust the pH of the medium to 5.7 with concentrated hydrochloric acid (HCl). Then sterilize the medium by sterile filtration or autoclaving.

[0266] Example 2: Preparation of OS-medium for isolating lactobacilli:

[0267] Table 2: OS culture medium

[0268]

[0269]

[0270] The pH of the culture medium was adjusted to 5.5 with concentrated hydrochloric acid (HCl). The culture medium was then autoclaved. 40 g / L of glucose was then added as a 10-fold dilution of the autoclaved medium.

[0271] Example 3: Rifampicin selection of lactobacilli:

[0272] Lactobacillus (L. diolivorans) DSM14421 was cultured overnight in a sterile glass culture dish in 5 ml of MRS medium (glucose + glycerol) + 0.03125 μg / ml of rifampicin and shaken at 30 ° C (180 rpm) in an anaerobic jar. This amount of rifampicin significantly inhibited the growth of lactobacillus. The cells were streaked on MRS agar plates (MRS medium + glucose + 1.5% agar) and cultured in an anaerobic jar at 30 ° C for 3 days. Single colonies were restreaked, cultured again on MRS agar plates, and then inoculated into liquid MRS medium (+ glucose) and plasmids were isolated using Quiagen DNeasy Blood & Tissue Kit (Cat. No. / ID: 69504). A clone (named Lactobacillus diolivorans Vogelbusch 1050, deposited in DSM 33056) was found to have a significantly different DNA pattern compared to the parental clone (DSM 14421), such as Figure 1 shown.

[0273] Example 4: Isolation of new Lactobacillus strains:

[0274] Six samples of corn silage, five samples of grass silage, two samples of 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, 30 g (dry weight) of each sample was suspended in a shake flask containing 100 ml of sterile 0.9% NaCl solution. After shaking for two minutes, the supernatant was diluted to 10% with sterile 0.9% NaCl solution.-9 Serial dilutions of the solution were made. 100 μL of each dilution was spread onto MRS and OS plates, both of which contained 50 mg / L vancomycin. The plates were incubated under anaerobic conditions for 3 days. After the incubation period, colonies were selected based on their morphological appearance. Only small colonies (1-4 mm) with clear edges, raised, smooth, shiny, opaque and non-pigmented were selected and re-streaked on the same plates from which they were isolated. The plates were incubated for 3 days in an anaerobic jar at 30°C with the addition of Oxoid from Thermo Scientific. TM AnaeroGen TM 2.5L sachets were then used for species analysis using a MALDI-TOF mass spectrometer (Bruker). Identification was performed immediately after cultivation to analyze fresh cultures and improve spectral quality. To prepare the HCCA matrix solution, 250 μL of standard solvent (OS solution) was added to an aliquot of undissolved HCCA matrix (Bruker, No. 8255344). The HCCA matrix was dissolved by vortexing at room temperature until the solution became clear. For identification, a small sample of a single colony was plated directly onto a spot on a MALDI target plate. The spot was overlaid with 1 μL of 70% formic acid (FA) and allowed to dry at room temperature. The spot was then overlaid with 1 μL of HCCA solution and allowed to dry at room temperature. The prepared target was then measured on the MALDI biotyper using the Bacteria Test Standard (BTS) format. Of the approximately 786 colonies analyzed, 579 could be identified, including four strains identified as Lactobacilli.

[0275] These strains were designated as Lactobacillus with a deposit number of DSM 33060, HM with a deposit number of DSM 33058, HH with a deposit number of DSM 33057, and HS with a deposit number of DSM 33059, respectively.

[0276] Example 5: Using Lactobacillus liolivorans) Vogelbusch 1050 will glucose Anaerobic co-fermentation with glycerol to produce 1,3-propylene glycol.

[0277] Table 3 shows the co-fermentation of glucose and glycerol to 1,3-propanediol (1,3-PD) using Lactobacillus vogelbusch 1050 (deposited under DSM 33056). The fermentation was carried out in a fed-batch process using a 5 mg / L vitamin B supplemented with 1% glycerol. 12 , 700 ml of MRS medium containing 30 g / L glucose and 10 g / L glycerol was used as batch culture medium (see Example 1), supplemented with 5 mg / L vitamin B 12A glucose / glycerol solution (0.1 mol glucose / 1 mol glycerol) with a glycerol concentration of 500 g / L was used as the feed medium. Feeding began after the glucose and glycerol were consumed from the batch culture (t = 20 h). Feed medium was added to the culture at an initial rate of 2.4 mL / h. The feed rate was continuously reduced according to Equation 1 until the process was complete (t = 188 h).

[0278] Feed [ml / h] = 0.0106 * t [h] - 2.61

[0279] Formula 1

[0280] The pH value was adjusted to 5.7 with 8 M sodium hydroxide (NaOH) throughout the process. Nitrogen (N2) was used for aeration (2 L / h) during the fermentation process to avoid oxygen contamination and carbon dioxide accumulation.

[0281] Table 3: Co-fermentation of glucose and glycerol to 1,3-propanediol (1,3-PD) using Lactobacillus Vogelbusch 1050 in a fed-batch process

[0282]

[0283] The yield of 1,3-propylene glycol was 62.8 wt% (g 1,3-propylene glycol / g total glycerol) and 70.0 wt% (g 1,3-propylene glycol / g used glycerol), and a titer of 81.1 g / L 1,3-propylene glycol was obtained. The maximum specific productivity was 7.2 g / L per gram of biomass.

[0284] Example 6: Using Lactobacillus liolivorans) Vogel Bushi 1050 co-fermented glucose and glycerol into 1,3-propanediol.

[0285] Table 4 shows the results of the co-fermentation of glucose and glycerol to convert glycerol to 1,3-propanediol (1,3-PD) using Lactobacillus vogelbusch 1050 (deposited under DSM 33056) without nitrogen sparging. The fermentation was carried out in a fed-batch process using a 5 mg / L vitamin B6 supplemented with 1% ethanol. 12 , 700 ml of MRS medium containing 30 g / L glucose and 10 g / L glycerol was used as batch culture medium (see Example 1), supplemented with 5 mg / L vitamin B 12A glucose / glycerol solution (0.1 mol glucose / 1 mol glycerol) with a glycerol concentration of 500 g / L was used as the feed medium. When the batch was started, the dissolved oxygen value was 80% (oxygen saturation was 100%). The dissolved oxygen concentration decreased continuously for 16 h to reach 0% (undetectable). Feeding was started after glucose and glycerol were consumed from the batch medium (t = 28 h). The feed medium was added to the culture at 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 8 M sodium hydroxide (NaOH). There was no purge gas in the culture.

[0286] The 1,3-propylene glycol yields were 62.4 wt% (g 1,3-propylene glycol / g total glycerol) and 69.6 wt% (g 1,3-propylene glycol / g glycerol used), with a titer of 80.4 g / L. The maximum specific productivity was 7.9 g / L per gram of biomass. These values ​​are very similar to those achieved in conventional processes using nitrogen purge (Example 5).

[0287] Table 4: Lactobacillus Vogelbusch 1050 in a fed-batch process without nitrogen purge Glucose and glycerol are co-fermented to convert glycerol into 1,3-propanediol (1,3-PD).

[0288]

[0289] Surprisingly, the strain grew well under aerobic conditions and without any nitrogen purge, producing 1,3-propanediol. Titers and yields were comparable (80.4 g / L compared to 81.1 g / L with a nitrogen purge; 62.4% compared to 62.8%). Particularly surprising was that the maximum specific productivity without a nitrogen purge, 7.9 g / L per gram of biomass, was higher than the maximum specific productivity with a nitrogen purge (7.2 g / L per gram of biomass).

[0290] Example 7: Lactobacillus liolivorans) to different sugars and glycerol Co-fermentation

[0291] Table 5 shows the co-fermentation of glucose, xylose, fructose, arabinose, mannose or sucrose with glycerol using the newly isolated lactobacillus strains. The fermentations were carried out in batches in glass eprouvettes using a 5% ethanol solution supplemented with 0.005 mg / L vitamin B6. 12, 2 ml of MRS medium containing 30 g / L of the corresponding sugar and 10 g / L of glycerol was used as the batch culture medium (see Example 1). The eprouvettes were shaken in an anaerobic jar at 30°C for 72 h, and the culture supernatant was analyzed by HPLC. The anaerobic jar was supplemented with Oxoid from Thermo Fisher Scientific. TM AnaeroGen TM 2.5L sachet.

[0292] Table 5: Concentration of 1,3-propanediol after 72 h of fermentation on MRS medium with the indicated carbon sources

[0293]

[0294]

[0295] All strains produced 1,3-propanediol with equal efficiency on glucose and glycerol. Interestingly, significant differences were observed on sucrose and glycerol. While Vogelbusch 1050 (deposited under DSM 33056), HF (deposited under DSM 33060), and HM (deposited under DSM 33058) were less efficient, strains HS (deposited under DSM 33059) and especially HH (deposited under DSM 33057) also produced 1,3-propanediol efficiently on sucrose and glycerol. Strain HS (deposited under DSM 33059) was particularly efficient on xylose and mannose. All strains grew on arabinose, but they did not accumulate significant amounts of 1,3-propanediol on arabinose or glycerol.

Claims

1. A method for producing a cellular 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 cellular metabolite, and isolating the cellular metabolite from the cell culture; wherein the lactobacillus cells are a strain of Lactobacillus deposited with DSM 33056; wherein the aerobic reactor system does not employ means for shielding the cell culture from oxygen; wherein the carbon source contained in the feed medium comprises a combination of glycerol and at least one of glucose, xylose, fructose, sucrose or mannose; and wherein the cellular metabolite is 1,3-propanediol. 2 . The method according to claim 1 , wherein the carbon source comprises glycerol having a purity lower than technical grade and an ash content of at least 0.1% (w / w).

3. The method according to claim 1, wherein the production phase is performed in fed-batch mode or continuous mode.

4. The method of claim 1, wherein the feed medium is continuously added to the cell culture during the production phase.

5. The method of claim 1, wherein prior to the production phase, the cell culture is carried out in batch mode using a growth medium to accumulate biomass.

6. A lactobacillus strain having a deposit number of DSM 33056.

7. Use of lactobacillus cells in cell culture for bioconverting a carbon source into a cellular metabolite by an aerobic fed-batch fermentation process, wherein the lactobacillus cells are a lactobacillus strain with a deposit number of DSM 33056; wherein the carbon source comprises glycerol; and the cellular metabolite is 1,3-propanediol.

8. Use of lactobacillus cells in cell culture for bioconverting a carbon source into a cellular metabolite via an aerobic fed-batch fermentation process, wherein the lactobacillus cells are a lactobacillus strain with a deposit number of DSM 33056; wherein the carbon source comprises a combination of glycerol and at least one of glucose, xylose, fructose, sucrose, or mannose; and the cellular metabolite is 1,3-propanediol.

9. Use according to claim 7 or 8, wherein the carbon source used for the bioconversion is glycerol with a purity lower than technical grade and an ash content of at least 0.1% (w / w).

10. Use according to claim 9, for the bioconversion of glycerol into 1,3-propanediol.

11. The use according to claim 10, wherein the glycerol is crude glycerol derived from biodiesel.

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