Method of preparing 1,3-propanediol

Abstract

This disclosure concerns methods of preparing 1,3-propanediol (1,3-PD) from glycerol using genetically engineered Lentilactobacillus diolivorans and Escherichia coli cells. The L. diolivorans is engineered to overexpress a glycerol transporter, glycerol dehydratase with its activation factor, and / or 1,3-propanediol oxidoreductase, while the E. coli is engineered to overexpress a 1,3-propanediol oxidoreductase. In one method of culture, the L. diolivorans is cultivated in glycerol-containing medium to produce 1,3-PD and the intermediate 3-hydroxypropionaldehyde (3-HPA). The E. coli is then grown in the L. diolivorans culture medium to convert the 3-HPA to 1,3-PD. Alternatively, the two types of cells may be cultured together in the presence of glycerol to produce 1,3-propanediol.

Description

[0001] METHOD OF PREPARING 1,3-PROPANEDIOL

[0002] Technical field

[0003] The present invention relates, in general terms, to methods of preparing 1,3 -propanediol. In particular, the specification teaches a method of preparing 1,3-propanediol from glycerol using bacteria.

[0004] Background

[0005] Synthetic polymers arc the cornerstone of modem life, underpinning a multitude of essential products such as plastics, fibers, and adhesives. Their omnipresent and versatile nature position them as indispensable elements across various industries and everyday applications. However, many synthetic polymers are made from fossil fuels, which are not sustainable. Recently, there has been more focus on developing biotechnological methods to produce polymers from renewable resources. Polytrimethylene terephthalate (PTT) is a polymer which finds extensive use in the food, pharmaceuticals, cosmetics and lubricant industries. PTT is synthesised from a 1,3-propanediol (1,3-PD) precursor, which can be produced biologically from a variety of carbon sources, among which glycerol is an attractive feedstock due to the relatively simple metabolism pathway from glycerol to 1,3-PD, and the abundance of low-cost crude glycerol as a byproduct from the biofuel industry. For example, roughly 10 tons of crude glycerol are generated for every 100 tons of biodiesel produced via transesterification. In addition, the intermediate 3-hydroxypropionaldehyde (3-HPA) is produced during glycerol metabolism, which is itself a highly sought-after specialty chemical due to its antimicrobial activity. Nonetheless, only a few microorganisms arc known to efficiently convert glycerol to 1,3-PD, and current yields of 1,3-PD from biotransformation arc very low. Therefore, it would be desirable to overcome or ameliorate the above-described problems, or at least to provide a useful alternative.

[0006] Summary

[0007] Disclosed herein is a method of preparing 1,3-propanediol from glycerol, the method comprising: a) cultivating Lentilactobacillus diolivorans cells in a cell culture in the presence of glycerol to produce 3-hydroxypropionaldehyde (3-HPA) and / or 1,3-propanediol (1,3-PD), wherein the Lentilactobacillus diolivorans cells arc genetically engineered to overexpress a glycerol transporter; and b) cultivating Escherichia coli cells in cell culture media from step a) to produce 1,3 -propanediol, wherein the Escherichia coli cells are genetically engineered to overexpress a 1 ,3-propanediol oxidoreductase.

[0008] Disclosed herein is a method of preparing 1,3-propanediol from glycerol, the method comprising cultivating Lentilactobacillus diolivorans cells and Escherichia coli cells in the presence of glycerol, wherein the L. diolivorans is genetically engineered to overexpress a glycerol transporter, and wherein the E. coli is genetically engineered to overexpress a 1,3- propanediol oxidoreductase.

[0009] Disclosed herein is a kit for biological production of 1,3-propanediol from glycerol, the kit comprising Lentilactobacillus diolivorans cells genetically engineered to overexpress a glycerol transporter, and Escherichia coli cells genetically engineered to overexpress a 1,3- propanediol oxidoreductase.

[0010] Brief description of the drawings

[0011] Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:

[0012] Figure 1. Plasmid maps for gene overexpression in L. diolivorans. (a) Plasmid map of pGAP / eg / pF for glycerol transporter, (b) Plasmid map of pGAP / hgZpF for glycerol transporter, (c) Plasmid map of pGAP / pduF for glycerol transporter, (d) Plasmid map of pGAPipduFCDEGH for glycerol transporter and B 12-dependant glycerol dehydratase and its reactiving factors, (e) Plasmid map of pGAP IpduF / dhBl B2 for glycerol transporter and B 12-independent glycerol dehydratase and its activating factor, (f) Plasmid map of pGAP!pduF / dhBIB2 / pduQ for glycerol transporter, B 12-independent glycerol dehydratase and its activating enzyme, combined with 1,3-PD oxidoreductase.

[0013] Figure 2. Modular whole-cell biotransformation for 1,3-PD production using engineered L. diolivorans and E. coli recombinants, (i) Glycerol metobolic pathway in gene ovcrcxprcssionL. diolivorans recombinant, (ii) Bioconversion of 3-HPA into 1,3-PD in 1,3- PD oxidoreductase overexpression E. coli recombinant.

[0014] Figure 3. The production of 3-HPA and 1 ,3-PD in different L. diolivorans strains and optimization fermentation condition for the first step whole-cell biotransformation with LMG199668 / / ?<7MF. (a) The production of 3-HPA and 1,3-PD in wild type strain L. diolivorans LMG 199668 and 3 L. diolivorans recombinants. LMG19668 / EV is a control strain which was transformed with empty vector, (b) The production of 3-HPA and 1,3-PD in LMG1996($ / pduF with different biotransformation time, (c) The production of 3-HPA and 1,3-PD in LMG199668 / p<7wF supplemented with varying glycerol concentrations in cell growth phase.

[0015] Figure 4. Module 11 development: SDS-PAGE analysis of 1,3-PD oxidoreductases (PDORs) from different microorganisms expressed in E. coli BL21 (DE3). Lane M: protein marker; lane 1: PDOR from Clostridium butyricum DSM5478; Lane 2: PDOR from Lentilactobacillus diolivorans; Lane 3: PDOR from Clostridium butyricum VPI1718; Lane 4: PDOR from Clostridium pasteurianum; Lane 5: PDOR from Klebsiella pneumoniae; Lane 6: PDOR from Citrobacter freundii.

[0016] Figure 5. The production of 3-HPA and 1,3-PD in the two modules and the reuse of the cells, (a) The production of 3-HPA and 1,3-PD after the first step whole-cell biotransformation with L. diolivorans recombinant and after second step whole-cell biotransformation with E. coli recombinants, (b) Test reuse of the cells of L. diolivorans recombinant and E. coli recombinant for 3-HPA and 1,3-PD production.

[0017] Figure 6. The HPLC chromatograms of culture media from the first step and second step whole-cell biotransformation, (a) The HPLC chromatogram of the first step whole-cell biotransformation with LMG196684?<7MF. (b) The HPLC chromatogram of the second step whole-cell biotransformation with BL-dhaT.

[0018] Figure 7. Module I development: 1,3-PD production by stepwise engineered L. diolivorans strains and fermentation optimization of the selected optimal producer, (a) 1 ,3-PD production by stepwise engineered L. diolivorans strains, (b) Enhanced 1,3-PD production by the selected optimal strain (Lac / 4) after response surface optimization. Figure 8. The concept of modular co-cultivation system, in which Module I consists of an L. diolivorans recombinant and Module II consists of an E. coll recombinant.

[0019] Figure 9. Optimization of co-cultivation systems: combining fixed concentration of optimal L. diolivorans strain from Module I (5 g / L) with varying ratios of engineered E. coli strains from Module II. Co-cultivation performance with the optimal strain from Module I (Lac / 4, 5 g / L) and engineered E. coli from Module II, where E. coli comprises: (a) 25% of total biomass, (b) 50% of total biomass, (c) 75% of total biomass, (d) Enhanced 1,.3-PD production and reduced 3-HPA accumulation at 1:1 strain ratio compared to wild-type LMG19668.

[0020] Detailed description

[0021] The present specification teaches compositions and methods for preparing 1,3-propancdiol (1,3-PD) from glycerol using genetically engineered Lentilactobacillus diolivorans and Escherichia coli as whole cell biocatalysts. The L. diolivorans is genetically engineered to overexpress a glycerol transporter to increase glycerol uptake, and may also be engineered to overexpress a glycerol dehydratase with its activation factor, and / or a 1,3-propanediol oxidoreductase (PDOR). The E. coli is engineered to overexpress a 1,3-propanediol oxidoreductase (PDOR). Glycerol dehydratase converts glycerol to an intermediate compound 3-hydroxypropionaldchydc (3-HPA), while 1,3-propancdiol oxidoreductase converts 3-HPA to 1,3-PD (Figure 2).

[0022] Although certain strains of L. diolivorans natively express glycerol metabolic pathway enzymes, conversion of glycerol to 1,3-PD can be inefficient in these strains and limited by accumulation of toxic 3-HPA in the culture. The inventors found that engineering L. diolivorans to overexpress a glycerol transporter, glycerol dehydratase and / or 1,3- propanediol oxidoreductase can improve glycerol biotransformation. Furthermore, engineered E. coli strains that overexpress 1,3-propanediol oxidoreductase can be used in conjunction with the engineered L. diolivorans to efficiently convert the 3-HPA intermediate to further increase the yield of 1 ,3-PD. The inventors show that the engineered L. diolivorans and E. colt cells may be cultured sequentially or together. Thus, in one example, the L. diolivorans may be cultured first in the presence of glycerol to produce a conditioned culture containing 3-HPA and / or 1,3-PD, and the E. coli is then grown in the conditioned culture to produce 1 ,3-PD. Alternatively, the L. diolivorans and the E. coli may co-cultured in the presence of glycerol to produce 1,3- PD. The combination of increased glycerol uptake and glycerol metabolism in the engineered L. diolivorans and the heterologous 1,3-PD production pathway in the engineered E. coli creates a synergistic system for high-yield 1,3-PD biosynthesis from glycerol. The inventors were able to achieve significant improvements in 1,3-PD yields over current bioproduction methods, with yields exceeding 28 g / L.

[0023] General definitions

[0024] As used herein, a “glycerol transporter” refers to a membrane-associated protein that facilitates glycerol uptake into a cell. The transporter may facilitate unidirectional transport of glycerol into the cell, or bidirectional transport of glycerol into and out of the cell.

[0025] As used herein, “glycerol dehydratase” refers to an enzyme which catalyses the conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA). The enzyme may be a multimeric protein containing, for example, two, three or more subunits. Glycerol dehydratases may be dependent on adenosylcobalamin (vitamin B 12) as a cofactor, or can exist in B 12- independent forms. Glycerol dehydratases may become inactivated over the course of catalysis, for example by the glycerol substrate or by exposure to oxygen. Co-expression of an “activating factor” or “activase” can help to restore enzyme activity. The gene for activating factors are frequently found alongside the gene for the glycerol dehydratase.

[0026] As used herein, “1,3-propanediol oxidoreductase” refers to an enzyme which catalyses the reduction of 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD), typically converting it. The enzyme is also known as 1,3-propanediol dehydrogenase.

[0027] The term “overexpression” as used herein with respect to a host cell, specifically a recombinant host cell, is intended to encompass increasing the expression of a polypeptide or protein (such as enzymes used in the biotransformation process) to a level greater than the cell normally produces. It is intended that the term encompass overexpression of homologous (endogenous), as well as heterologous sequences or proteins, specifically aiming at the increase of the productivity of the cell to produce a product, e.g., at least 1.5- fold, preferably at least 2-fold, more preferred at least 3-fold as compared to a wild-type host cell of the same type.

[0028] “Expression vectors” or “vectors” used herein are DNA sequences that are required for the transcription of cloned recombinant nucleotide sequences, i.e. of recombinant genes and the translation of their mRNA in a suitable host organism. Such expression vectors usually comprise an origin for autonomous replication in the host cells, selectable markers (e.g. an amino acid synthesis gene or a gene conferring resistance to antibiotics such as erythromycin, chloramphenicol, zeocin, kanamycin, G418 or hygromycin), a number of restriction enzyme cleavage sites, a suitable promoter sequence and a transcription terminator, which components are operably linked together. The terms “plasmid” and “vector” as used herein include autonomously replicating nucleotide sequences as well as genome integrating nucleotide sequences.

[0029] The term “genetically engineered” as used herein refers to a recombinant organism that is used for producing a fermentation product. The organism is typically a production cell line engineered to improve a production process or to enable the production of a new product. The term is understood in contrast to the “wild-type” organism, which is typically not genetically engineered.

[0030] The term “recombinant” as used herein means “being prepared by or the result of genetic engineering”. Thus, a “recombinant microorganism” comprises at least one “recombinant nucleic acid”. A recombinant microorganism may be a mutant produced by a suitable method mutagenesis, and / or specifically comprises an expression vector or cloning vector, or it has been genetically engineered to contain a recombinant nucleic acid sequence or recombinant metabolic pathway to produce cell metabolites at high yield or to produce novel cell metabolites which the wild-type cell (before recombination) would not produce in significant amounts.

[0031] The term “cell line” as used herein shall refer to an established clone of a particular cell type that has acquired the ability to proliferate over a prolonged period of time. The term “host cell line” refers to a cell line as used for expressing an endogenous or recombinant gene or products of a metabolic pathway to produce polypeptides or cell metabolites mediated by such polypeptides. A “production host cell line” or “production cell line” is commonly understood to be a cell line ready-to-use for cultivation in a bioreactor to obtain the product of a production process. The applicable production cell line may produce a number of useful intermediates and products by fermenting a low molecular weight sugar produced by saccharifying the treated biomass materials. The applicable production cell line may also produce the useful intermediates and products while saccharifying the treated biomass materials by producing the necessary enzymes. For example, fermentation or other bioprocesses can produce alcohols, organic acids, hydrocarbons, hydrogen, proteins or mixtures of any of these materials.

[0032] In general, the recombinant nucleic acids or organisms as referred to herein may be produced by recombination techniques well known to a person skilled in the art. In accordance with the present invention, conventional methods of molecular' biology, microbiology, and recombinant DNA techniques within the skill of the art may be employed. Such techniques are explained in detail e.g., in Maniatis, Fritsch & Sambrook, Molecular Cloning: A Laboratory Manual (1982).

[0033] The term “cell culture” or “culturing” or “cultivation” as used herein refers to the maintenance of the bacterial cells in an artificial, e.g., an in vitro environment, under conditions favouring growth, differentiation, or continued viability, in an active or quiescent state, of the cells, specifically in a controlled biorcactor according to methods known in the industry.

[0034] When culturing a cell culture using appropriate culture media, the cells are brought into contact with the media in a culture vessel or with substrate under conditions suitable to support culturing the cells in the cell culture. As described herein, a culture medium is provided that can be used for the growth of cells. Standard cell culture techniques are well- known in the art.

[0035] The term “conditioned culture” herein refers to a culture that has been exposed to cells for a period of time, such as hours, days or longer, and which contains metabolites and other metabolic by-products produced by the cells. “Conditioned medium” refers to culture medium isolated from a conditioned culture. The term “sequence identity” as used herein indicates that two or more nucleotide sequences have (to a certain degree, up to 100%) the same or conserved base pairs at a corresponding position. "Percent (%) identity" with respect to the nucleotide sequence of a gene or genome is defined as the percentage of nucleotides in a candidate DNA sequence that is identical with the nucleotides in the DNA sequence to which the candidate sequence shall be compared, after aligning the sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotide sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0036] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0037] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

[0038] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0039] Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase “consisting essentially of’, and variations such as “consists essentially of’ will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined. L. diolivorans

[0040] Strains of L. diolivorans that can be used include wild-type and deposited strains such as LMG19668, G77, DSM14421, DSM14424, DSM33056, DSM33057, DSM33058, DSM33059, DSM33060, or any derivatives thereof. These strains can be obtained from, e.g., the Belgian Coordinated Collections of Microorganisms (BCCM), or the Leibniz Institute DSMZ-Gcrman Collection of Microorganisms and Cell Cultures. In one embodiment, the L. diolivorans cells are L. diolivorans LMG199668 orL. diolivorans DSM14421. These strains possess a native pathway for 1,3-PD production, can directly utilise crude glycerol, have a Generally Regarded as Safe (GRAS) status, and have high stress tolerance during culture, and are advantageous for the methods herein.

[0041] L. diolivorans may be cultivated in any suitable media known in the ail for L. diolivorans culture. The medium may be a defined medium or a complex medium. For example, media commonly used for Lactobacillus culture, such as MRS medium, M17 medium or clostridial medium, may be used for L. diolivorans cultivation. Also contemplated herein are media derived from agricultural or industrial byproducts, such as whey-based formulations and formulations containing hydrolysates of starchy plant material or hydrolysates of lignocellulosic plant material.

[0042] The L. diolivorans medium contains glycerol as raw material for bioconversion to 3-HPA and 1,3-PD. The medium may contain one or more additional carbon sources such as glucose, xylose, arabinose, fructose, sucrose, lactose or others to support growth of the bacteria.

[0043] In some embodiments, the L. diolivorans cells are cultivated in the presence of about 0.5% w / v to about 3% w / v glycerol, such as about 0.5% w / v, about 1% w / v, about 1.5% w / v, about 2% w / v, about 2.5% w / v, or about 3% w / v glycerol. The inventors have found that glycerol concentrations higher than about 3% w / v can lead to reduced yields of both 3-HPA and 1,3- PD.

[0044] The glycerol may be crude or refined glycerol. Crude glycerol is generated by a number of commercial and experimental processes, including saponification, e.g., hydrolysis of triacylglyccrol to produce fatty acids and glycerol, and biodiesel production, in which transesterification of fats and oils occurs to produce methyl esters and a glycerol byproduct. Crude glycerol typically contains contaminants and / or impurities. The removal of these contaminants and / or impurities using processes known in the art produces refined glycerol. Refined glycerol may be sold as a product that is, e.g., at least 99.5% pure.

[0045] The L. diolivorans medium may be supplemented with vitamin B12. Vitamin B 12 is a cofactor for B12-dcpcndcnt glycerol dehydratases, and although the vitamin is present in most microbial complex media, B 12 supplementation can improve the efficiency of glycerol bioconversion, in particular where the L. diolivorans is engineered to overexpress a B 12- dependent glycerol dehydratase. In one embodiment, vitamin B 12 is provided at a concentration of about 5 mg / ml in the L. diolivorans medium.

[0046] L. diolivorans may be cultivated at a temperature in the range of about 25°C to about 40°C. Different culture temperatures may be used for protein expression and biotransformation. For example, the L. diolivorans may be cultured at an initial lower temperature (such as temperatures below 30°C) for transporter or enzyme overexpression, and the culture temperature is then raised for more efficient biotran formation. Tn some embodiments, the L. diolivorans is cultivated at a temperature in the range of about 30°C to about 37°C for biotransformation. In one embodiment, the L. diolivorans is cultivated at a temperature of about 30°C for biotransformation.

[0047] L. diolivorans is a facultative anaerobe and may be cultured under aerobic or anaerobic conditions. In one embodiment, the L. diolivorans is cultured anaerobically when in monoculture and aerobically when in co-culture with E. coli. In one embodiment, the L. diolivorans is cultured anaerobically both in monoculture and in co-culture with the E. coli. In one embodiment, the L. diolivorans is cultured anaerobically during the growth phase and aerobically during the production phase. In one embodiment, the L. diolivorans is cultured anaerobically for both the growth phase and the production phase.

[0048] E. coli

[0049] E. coli strains suitable for industrial production are well known in the art. These include E. coli K-12 derivatives such as MG1655 and W3110, B strains such as BL21 and C41, and E. coli W.

[0050] E. coli may be cultivated in any suitable culture media, including both defined and complex media. Complex media may include, for example, LB broth, Tryptic Soy Broth, Terrific Broth, 2xYT, and the like. Also contemplated herein are media derived from agricultural or industrial byproducts, such as formulations containing hydrolysates of starchy plant material or hydrolysates of lignocellulosic plant material.

[0051] E. coli may be cultivated at a temperature in the range of about 15°C to about 40°C. Different culture temperatures may be used for protein expression and biotransformation. For example, the E. coli may be cultured at an initial lower temperature (such as temperatures below 20°C) for transporter or enzyme overexpression, and a higher culture temperature is then used for biotransformation. In some embodiments, the E. coli is cultivated in the range of about 30°C to about 37°C for biotransformation. In one embodiment, the E. coli is cultivated al a temperature of about 30°C for biotransformation.

[0052] E. coli is a facultative anaerobe and may be cultured under aerobic or anaerobic conditions. In one embodiment, the E. coli is cultured aerobically when in monoculture and anaerobically when in co-culture with L. diolivorans. In one embodiment, the E. coli is cultured aerobically both in monoculture and in co-culture with the L. diolivorans. In one embodiment, the E. coli is cultured aerobically during the growth phase and anaerobically during the production phase. In one embodiment, the E. coli is cultured aerobically during both the growth and the production phases.

[0053] Glycerol transporters

[0054] In preferred embodiments, the L. diolivorans is engineered to overexpress a glycerol transporter to increase uptake of glycerol for biotransformation.

[0055] The glycerol transporter may be a bacterial glycerol transporter. The glycerol transporter may, for example, be derived from Lentilactobacillus diolivorans, Escherichia coli or Bacillus suhtilis. In some embodiments, the glycerol transporter is derived from L. diolivorans. In one embodiment, the L. diolivorans is engineered to overexpress a native glycerol transporter. In one embodiment, the L. diolivorans is engineered to overexpress a glycerol transporter derived from L. diolivorans DSM14421 or LMG19668.

[0056] In one embodiment, the glycerol transporter comprises an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity) to an amino acid sequence in SEQ ID NO: 1.

[0057] The overexpression of the glycerol transporter may increase uptake of glycerol into the engineered L. diolivorans cells compared to wild-type L. diolivorans. Thus, in some embodiments, the genetically engineered L. diolivorans cells are characterised by an increased uptake of glycerol compared to wild-type L. diolivorans cells. For example, the engineered L. diolivorans may exhibit an increase in glycerol uptake that is at least 10%, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 2-fold, about 3-fold, about 4-fold, about 5-fold, or more than 5-fold, compared to the wild-type L. diolivorans cells from which they were engineered.

[0058] Glycerol dehydratases

[0059] In some embodiments, the L. diolivorans is engineered to overexpress a glycerol dehydratase and a glycerol dehydratase activation factor to increase conversion of glycerol to 3-HPA. The glycerol dehydratase may be a bacterial glycerol dehydratase.

[0060] In some embodiments, the glycerol dehydratase is a bacterial B 12-dependent glycerol dehydratase. In one embodiment, the glycerol dehydratase is a B 12-dependent glycerol dehydratase derived from Lentilactobacillus diolivorans .

[0061] In one embodiment, the B12-dcpcndcnt glycerol dehydratase comprises a polypeptide comprising an amino acid sequence having at least 70%> sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity) to an amino acid sequence in SEQ ID NO: 3, 5 or 7. In some embodiments, the glycerol dehydratase is a bacterial B12-independent glycerol dehydratase. In some embodiments, the glycerol dehydratase is a B12-independent glycerol dehydratase derived from Clostridium butyricum or Lentilactobacillus diolivorans. The expression of a B12-independent glycerol dehydratase and its activation factor can be advantageous in enabling efficient glycerol-to-3-HPA conversion without requiring vitamin B 12 supplementation during culture.

[0062] In some embodiments, the B 12-independent glycerol dehydratase comprises a polypeptide comprising an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity) to an amino acid sequence in SEQ ID NO: 13.

[0063] In some embodiments, the glycerol dehydratase activation factor comprises a polypeptide comprising an amino acid sequence having at least 70% sequence identity to an amino acid sequence in SEQ ID NO: 9, 11 or 15.

[0064] The overexpression of the glycerol dehydratase may increase production of 3-HPA by the genetically engineered L. diolivorans cells compared to wild-type L. diolivorans. Thus, in some embodiments, the genetically engineered L. diolivorans cells are characterised by an increased production of 3-HPA compared to wild-type L. diolivorans cells. For example, the engineered L. diolivorans may exhibit an increased production of 3-HPA that is at least 10%, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 2-fold, about 3-fold, about 4-fold, about 5-fold, or more than 5-fold, compared to the wild-type L. diolivorans cells from which they were engineered.

[0065] 1 ,3-propanediol oxidoreductases

[0066] In preferred embodiments, the E. coli is engineered to overexpress a 1,3-propanediol oxidoreductase to reduce intracellular accumulation of 3-HPA and increase conversion of 3- HPA to 1,3-PD. In some embodiments, the L. diolivorans is also engineered to overexpress a 1,3- propanediol oxidoreductase to increase conversion of 3-HPA to 1,3-PD. The 1,3 -propanediol oxidoreductase may be a bacterial 1,3-propanediol oxidoreductase. The 1,3 -propanediol oxidoreductase may, for example, be derived from Clostridium bulyricum, Lentilactobacillus diolivorans, Citrobacter freundii, Klebsiella pneumoniae, or Clostridium pasleurianum.

[0067] In some embodiments, the 1,3-PD oxidoreductase comprises an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity) to an amino acid sequence in SEQ ID NO: 17, 19, 21, 23, 25 or 27.

[0068] The overexpression of the 1,3-PD oxidoreductase may increase production of 1,3-PD by the genetically engineered L. diolivorans or E. coli cells compared to wild-type L. diolivorans or E. coli cells. Thus, in some embodiments, the engineered L. diolivorans or E. coli is characterised by an increased production of 1,3-PD compared to wild-type L. diolivorans or E. coli cells. For example, the engineered L. diolivorans or E. coli may exhibit an increased production of 1 ,3-PD that is at least 10%, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 2-fold, about 3-fold, about 4-fold, about 5-fold, or more than 5-fold, compared to the wild-type L. diolivorans or E. coli cells from which they were engineered.

[0069] Expression vectors

[0070] The genetically engineered cells of this disclosure may contain expression constructs encoding the glycerol transporter or glycerol-metabolising enzyme. The expression construct can be stably integrated into the host cell genome or replicated as an episomal vector, such as a plasmid.

[0071] Suitable vectors will be those which are compatible with the bacteria employed. Expression vectors suitable for use in E. coli are well known in the art. For L. diolivorans, it may be preferred to use a plasmid originating from wild-type L. diolivorans as an expression vector. Alternatively, a plasmid containing a L. diolivorans origin of replication may be used. Protocols for obtaining and using such vectors are known to those in the art and may be referenced in Sambrook ct al.. Molecular Cloning: A Laboratory Manual — volumes 1, 2, 3 (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1989).

[0072] Codons in the expression construct may be optimised for a particular expression host, as is well known in the art. Genes in the expression construct may be operably linked to one or more regulator}' sequences. Examples of regulatory sequences include promoters, operators, enhancers, ribosomal binding sites, and sequences that control transcription and translation initiation and termination. The regulator}' sequences may be operably linked to the DNA sequence to be expressed. For example, a promoter sequence is said to be operably linked to a coding sequence, if the promotor controls the transcription of the coding sequence.

[0073] Suitable promoter sequences for use with bacterial host cells herein may include but are not limited to promoters obtained from Lactobacillus sp. , Lactococcus sp. , Staphylococcus sp. , Pediococcus sp., Enterobacteria sp., Streptococcus sp., Oenococcus sp., or E. coli. The promoter is not limited to any particular species provided that they can function in the bacterial host cell. The promoter can be constitutive or regulatable. The regulatable promoters can be, for example, inducible. The promoter sequences can be derived, for example, from the host cell, from another organism, or can be synthetically derived.

[0074] Any desired promoter can be used to regulate the expression of the inserted genes. For example, promoters may be induced by nutrient source, nutrient starvation, growth phase, heat shock, cold shock, oxidative stress, salt stress, environmental stress, metal concentration, specific metabolites or organic compounds, light exposure, etc. In other embodiments, the gene(s) may be under the transcriptional control of a constitutive promoter. In this way, a sustained level of transcription and, therefore, enzymatic activity of the corresponding protein can be maintained during the whole period of culture. The constitutive promoter may be endogenous to the host cell. This has the advantage that no recombinant transcription factor has to be present in the host cell. The endogenous promoter is usually well-recognised by the host cell without the need to introduce further genetic modifications.

[0075] Vectors or expression constructs may be transferred into the host cell by transformation. Preferred methods of transformation for the uptake of the recombinant DNA by bacteria include chemical transformation, electroporation or transformation by protoplastation. Transformants can be obtained by introducing such a vector, c.g. a plasmid, into a host and selecting transformants which express the relevant protein or host cell metabolite with high yields. The polypeptides encoded by the genes can be produced using the recombinant host cell line by culturing a transformant, thus obtained in an appropriate medium, isolating the expressed product or metabolite from the culture, and optionally purifying it by a suitable method.

[0076] Modes of culture

[0077] The cell cultures as described herein particularly employ techniques which provide for the production of cell metabolites of interest by biotransformation, such as to obtain the product in the cell culture medium, which is separable from the cellular biomass, herein referred to as “cell culture supernatant”, and may be isolated and optionally purified to obtain the product at a higher degree of purity.

[0078] Cell culture media provide the nutrients necessary to maintain and grow cells in a controlled, artificial, and in vitro environment. Characteristics and compositions of the cell culture media may vary depending on the particular cellular requirements. Important parameters include osmolality, pH, and nutrient formulations. Feeding of nutrients may be done in a continuous or discontinuous mode according to methods known in the art.

[0079] Growth and / or production can suitably take place in batch mode, fed-batch mode or continuous mode. A preferred embodiment includes a batch culture to accumulate biomass followed by a fed-batch culture for efficient bioconversion.

[0080] Substrates or raw materials for bioconversion (such as glycerol or 3-HPA) may be contained in the feed of a fed-batch process, e.g., under substrate-limited conditions, i.e., using limited amounts of the substrate to keep a production cell line under growth-limited conditions and in the production mode.

[0081] Whereas a batch process is a cell culture mode in which all the nutrients necessary for culturing the cells are contained in the initial culture medium, without additional supply of further nutrients during fermentation, in a fed-batch process, after a batch phase, a feeding phase takes place in which one or more nutrients are supplied to the culture by feeding. Although in most processes the mode of feeding is critical and important, the cell culture and methods described herein are not restricted with regard to a certain mode of cell culture.

[0082] In one embodiment, host cells described herein are cultured in a continuous mode. A continuous fermentation process is characterized by a defined, constant, and continuous rate of feeding of fresh culture medium into a bioreactor, whereby culture broth is at the same time removed from the bioreactor at the same defined, constant, and continuous removal rate. By keeping culture medium, feeding rate and removal rate at the same constant level, the cell culture parameters and conditions in the bioreactor remain constant.

[0083] L. diolivorans or E. coli culture may occur in two stages: a “growth phase” to accumulate cellular biomass, followed by a “production phase” during which biotransformation occurs. The growth and production phases may be conveniently performed in batch, fed-batch or continuous mode. The medium used for the growth phase can, but need not be, identical to the medium used during the production phase. For example, L. diolivorans may be grown on glucose or xylose for biomass accumulation followed by a feed containing glycerol and optionally vitamin B 12 for biotransformation.

[0084] The expression of the glycerol transporter and glycerol metabolising enzymes may be initiated during the growth phase. For example, where expression of the glycerol transporter, glycerol dehydratase and / or 1,3-PD oxidoreductase is under the control of an inducible promoter, an inducer may be added during the growth phase to induce protein expression prior to the production phase. Alternatively, the inducer may be added only during the production phase. In yet other embodiments, the inducer is added throughout the growth and production phases.

[0085] In some embodiments, the L. diolivorans is cultivated for at least 24 hrs (such as for about 24 hrs, about 30 hrs, about 36 hrs, or longer than 36 hrs) to accumulate biomass prior to sequential culture or co-culture with the E. coli cells.

[0086] In some embodiments, the E. coli is cultivated for at least 8 hrs (such as for about 8 hrs, about 12 hrs, about 16 hrs, about 24 hrs, or longer than 24 hrs) to accumulate biomass prior to sequential culture or co-culture with the L. diolivorans. (a) Sequential culture

[0087] In one embodiment, methods herein comprise sequential culture of the L. diolivorans and E. coli cells. The L. diolivorans cells may be cultivated first in the presence of glycerol to produce a conditioned culture containing 3-HPA and / or 1,3-PD, and the E. coli cells are then cultivated in the conditioned culture to produce 1,3-PD.

[0088] For sequential culture, the Lentilactobacillus diolivorans cells may be cultivated in the presence of glycerol for at least 3 hours to produce 3-HPA and 1,3-PD, such as for about 3 hr, about 4 hr, about 5 hr, or about 6 hr. This is found to provide sufficient time for accumulation of 3-HPA and 1,3-PD for the next stage of bioconversion.

[0089] Conditioned medium containing 3-HPA and 1,3-PD (i.e., culture supernatant) may be isolated from the L. diolivorans culture for use in the E. coli culture. The medium may be clarified to remove cells, debris and other particulates, and optionally pasteurised or sterilised prior to being used for E. coli culture. The conditioned medium may be supplemented to an E. coli culture (such as an E. coli culture which has undergone expansion to accumulate biomass) to initiate conversion of 3-HPA. Alternatively, the E. coli may be inoculated into the conditioned medium so that both cell growth and 3-HPA bioconversion occurs in the conditioned medium. In another embodiment, the conditioned medium is supplemented with additional media components before being used for E. coli culture. For example, the pH of the medium may be adjusted with suitable buffers, and additional carbon and / or nitrogen sources may be added to the medium prior to E. coli culture.

[0090] The method may comprise cultivating E. coli cells in the L. diolivorans conditioned medium for at least 6 hours, such as for about 6 hr, about 7 hr, about 8 hr, about 9 hr, about 10 hr, about 11 hr, about 12 hr, or longer than 12 hr.

[0091] (b) Co-culture

[0092] Methods herein may comprise co-culture of the genetically engineered L. diolivorans and E. coli cells in the presence of glycerol. Co-culture can be advantageous as it enables immediate consumption of the toxic intermediate 3-HPA by the E. coli cells. The L. diolivorans cells and the E. coli cells may be present at different ratios for the coculture. In some embodiments, the E. coli cells comprise at least about 25% of the cells in the co-culture. For example, the E. coli may comprise about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the co-culture. In one embodiment, the E. coli cells comprise about 50% of the cells in the co-culture.

[0093] In some embodiments, the amount of E. coli cells present in the co-culture is at least about 25% of the amount of L. diolivorans cells present. For example, the amount of E. coli cells present may be about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 1.2-fold, about 1.4-fold, about 1.6-fold, about 1.8-fold, about 2-fold, about 2.2-fold, about 2.4-fold, about 2.6-fold, about 2.8-fold, or about 3-fold of the amount of L. diolivorans cells present. In one embodiment, the amount of E. coli cells present in the co-culture is about the same as (i.e., about 100% of) the amount of L. diolivorans cells present.

[0094] The L. diolivorans cells may be co-cultured with the E. coli cells for at least 12 hrs for 1,3- PD production. For example, the L. diolivorans cells may be co-cultured with the E. coli cells for about 12 hrs, about 15 hrs, about 18 hrs, about 20 hrs, about 24 hrs, about 28 hrs, about 32 hrs, about 36 hrs, or longer than 36 hrs for 1,3-PD production. In one embodiment, the L. diolivorans cells is co-cultured with the E. coli cells for at least 18 hrs.

[0095] Co-culture may be performed at a temperature between 25°C to 37°C, and may be performed under aerobic or anaerobic conditions. In one embodiment, the co-culture is performed anaerobically at 30°C.

[0096] 1,3-PD isolation

[0097] The method may comprise isolating 1,3-propanediol from the cell culture of E. coli cells (for sequential culture), or from the co-culture of E. coli and L. diolivorans cells. The medium may be clarified to remove cellular matter, particulates and high molecular weight biomolecules prior to 1 ,3-PD extraction. Clarification may be performed using methods known to the skilled person, such as filtration, microfiltration, ultrafiltration, centrifugation and / or ion exchange chromatography. The medium may be subjected to an evaporation step to reduce the amount of water, for instance, to less than about 25% v / v to facilitate clarification and / or 1,3-PD isolation.

[0098] The 1 ,3-propanediol can be isolated and purified using techniques known in the art. Solvent extraction, distillation and chromatography are preferred, particularly for preparative isolation.

[0099] A highly purified product may be produced which is essentially free from contaminating proteins, and preferably has a purity of at least 90%, more preferred at least 95%, or even at least 98%, up to about 100%. The purified products may be obtained by purification of the cell culture supernatant or else from cellular debris.

[0100] The isolated and purified product can be identified by conventional methods such as high pressure liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS), NMR spectroscopy, infrared spectroscopy and Raman spectroscopy.

[0101] Kits

[0102] Disclosed herein is a kit for biological production of 1,3-propanediol from glycerol, the kit comprising Lentilactobacillus diolivorans cells genetically engineered to overexpress a glycerol transporter, and Escherichia coli cells genetically engineered to overexpress a 1,3- propancdiol oxidorcductasc.

[0103] In some embodiments, the L. diolivorans cells are further engineered to overexpress (i) a glycerol dehydratase and a glycerol dehydratase activation factor, and / or (ii) a 1 ,3- propanediol oxidoreductase.

[0104] The two bacterial strains may be provided separately or in a combined formulation, depending on the intended application. The bacteria cells may be provided in a sterile, stable and storable form. For example, the cells may be provided in freeze-dried (lyophilised) form containing suitable cryoprotectants (e.g., trehalose, sucrose, or skim milk) to preserve viability. The cells may also be provided preserved in a cryoprotective solution (e.g., 15- 25% w / v glycerol) and stored at ultra-low temperatures (e.g., -80°C). Such stocks may be thawed and cultured directly for experimental use. In other embodiments, cells may be provided as live cultures stored in semi-solid agar contained within sealed tubes. These cultures are suitable for transport at ambient temperatures and can be streaked directly onto solid media for propagation.

[0105] The kit may optionally comprise buffers, growth media and / or glycerol feedstocks for bacterial culture. Furthermore, the kit may include instructions for culturing conditions, growth media compositions, and parameters for induction of gene expression, if applicable.

[0106] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0107] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications, which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0108] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0109] Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

[0110] EXAMPLES

[0111] Materials and methods

[0112] Bacteria strains and plasmids E. coli BL21 (DE3) was used as a host strain for heterologous expression. E. coli DH5a was used as a host for plasmids propagation. Lenlilaclobacillus diolivorans LMG 19668 was used as a native producer of 1 ,3-PD. pET-28a (+) was used as expression plasmid. The DNA sequence eglpF of glycerol transporter was amplified from E. coli BL21 (DE3). The DNA sequence bglpF of glycerol transporter was amplified from Bacillus subtilis. The DNA sequence pduF of glycerol transporter was amplified from Lentilactobacillus diolivorans DSM 14421. The DNA sequences dhBlB2 of B12-indcpcndcnt glycerol dehydratase and its activation factor was ordered from Twist. The DNA sequence pduQ of 1,3-PD oxidoreductase was amplified from Lentilactobacillus diolivorans DSM 14421. All these genes were cloned into a vector carrying a replication origin of L. diolivorans (repA), under the control of GAP, PGM and P32 promoters of L. diolivorans as well as ampicillin and erythromycin resistance (Figure 1). In case of the empty vector (EV) control without introducing the glycerol transporter encoding gene.

[0113] Transformation procedure of L. diolivorans

[0114] An optimized transformation procedure was developed for L. diolivorans LMG 19668. In brief, 50 mL MRS medium supplemented with 2% (w / v) glucose was inoculated with L. diolivorans LMG 19668 cryo culture and incubated overnight at 30 °C without shaking. 50 mL MRS supplemented with 2% (w / v) glucose and 1% (w / v) glycine was inoculated with the overnight culture to ODeoo of 0.25AU and incubated at 30 °C for 3 h without shaking. Cells were harvested by centrifugation with 5000 g, 5 min, 4 °C and washed with 0.3 M sucrose with 4 times and adding 50 mM EDTA of pH 8.0 at the third time washing. The cells were finally resuspended in 100 pL electroporation buffer (272 mM sucrose, 7 mM sodium phosphate, 0.5 mM MgCh, pH 7.4). An 80 pL aliquot of cells was mixed with 3 pg DNA and electroporation was carried out at 2.5 KV, 200 , and 25 pF in an electroporation cuvette with 0.4 cm gap width. Immediately after electroporation, the cells were resuspended in 900 pL MRS supplemented with 2% (w / v) glucose, 0.3 M sucrose and 20 mM MgCh. This mixed culture was incubated at 30 °C, 180 rpm for 3 h. After regeneration, spread the culture on a plate supplemented with 5 pg / mL erythromycin to wait for colonies appear.

[0115] L. diolivorans culture Modified MRS medium containing 5 mg / mL vitamin B12, 5 pg / mL erythromycin, 3% glucose and 1% glycerol was used for L. diolivorans biomass generation. The bacteria were collected and used as whole cell biocatalysts to convert glycerol to 3-HPA and 1,3-PD.

[0116] E. coli culture

[0117] The plasmids pET28a-d / ia77, pET28a-d / ia7'2, pET28a-d / M73, pET28a-d / M7'4, pET28a- dhaT5 and pET28a-<f / ia76 were transferred into competent cells of E. coli BL21(DE3), and the recombinants were named E. coU / \ . E. colill, E. colU3, E. colUE E. colU5 and E. colitb, respectively. The recombinants were inoculated and cultivated in LB with kanamycin at 37°C. 200 pM IPTG was added to the medium when the OD600 was between 0.6-0.8, and the cells were cultured at 16°C overnight for enzyme expression. For SDS-PAGE, cells were centrifuged for 10 min at 3900 rpm, washed and resuspended in PBS buffer, then lysed by ultrasonication. The lysate was centrifuged to remove cell debris, and the supernatant was used for analysis.

[0118] The recombinant BL21 -d / ;«7 was used as whole cells biocatalysts for biotransformation of 3-HPA to 1 ,3-PD (Figure 2). BL21 -d / iaT were harvested after routinely grown in LB medium and induced by IPTG. Then the cells of BE21-dhaT were incubated in 100 mM potassium phosphate buffer (pH 6.0) at 30 °C for 30 min with 2% TritonX-100 (v / v) for permeabilization and washed twice with 100 mM potassium phosphate buffer (pH 6.0), then were used for the second step biotransformation.

[0119] Co-cultivation of Lentilactobacillus diolivorans and Escherichia coli

[0120] Co-culture was carried out in two stages: biomass production and glycerol biotransformation. L. diolivorans cells were cultured anaerobically at 30°C in MRS broth with 1 g / L glycerol and 30 g / L glucose. Engineered E. coli strains were grown in LB medium (50 mg / L kanamycin) at 37°C until OD600 reached 0.6, induced with 0.2 mM IPTG, and cultured for additional 12 h at 16°C. Cells were harvested by centrifugation (8000 x g, 4°C, 10 min), washed with potassium phosphate buffer (PPB, 0.1 M, pH 7.0). The collected cells of L. diolivorans and E. coli were resuspended in MRS (pH 7) to 10 g / L dry cell weight. Biotransformation was conducted at 30°C under anaerobic condition. Measurement of 3-HPA and 1,3-PD

[0121] 3-HPA was quantified by the colorimetric method or HPLC. Briefly, 1 mL of the appropriately diluted sample was mixed with 0.75 mL of 10 mM DL-tryptophan solution and 3 mL 37% HC1. 1,3-PD were determined with HPLC analysis (Agilent) with an Aminex HPX-87H column (300 mm x 7.8 mm; Biorad). The column was operated at 55°C and a flow rate of 0.8 mL / min with 5 mM H2SO4 as mobile phase. A refraction index detector (RID) was used for 3-HPA and 1,3-PD detection. The standards of 3- Hydroxypropionaldyhyde, 1,3-propanediol and Sulfuric acid were purchased from Sigma- Aldrich.

[0122] EXAMPLE 1: L. diolivorans engineering enhances 3-HPA and 1,3-PD production

[0123] Enhancement of 3-HPA production through upregulating glycerol uptake

[0124] 3-HPA serves as intermediate in the biosynthesis of 1,3-PD through glycerol metabolism pathway. Therefore, improving the efficiency of 3-HPA biosynthesis is essential for enhancing 1 ,3-PD production. The key stages in 3-HPA production involve the initial uptake of glycerol into the cell followed by its conversion to 3-HPA through glycerol dehydratase. Lactobacillus has been observed to accumulate 3-HPA outside the cell, keeping intracellular levels low and non-toxic, suggesting that export is not the limiting factor. Therefore, limitations associated with glycerol uptake and its subsequent conversion into 3-HPA was primarily investigated.

[0125] To enhance glycerol uptake in L. diolivorans LMG19668, the constitutive GAP promoter (glyceraldehyde- 3 -phosphate dehydrogenase) was utilized to overexpress three glycerol uptake facilitator proteins from different microorganisms: EglpF from E. coli BL21, BglpF from Bacillus subtilis, and pduF native to L. diolivorans DSM 14421, resulting in the generation of LMG19668 / eg / / ?L’, LMG 19668 / bglpF, and LMG19668 / pduL strains. For control purposes, an empty vector was introduced into L. diolivorans LMG19668, generating LMG19668 / EV strain. Subsequently, these strains were used as whole-cell biocatalysts for glycerol-to-3-HPA conversion, with glycerol as the substrate, over 24 h incubation. Compared to unmodified L. diolivorans LMG19668 and LMG19668 / EV, among these Lactobacillus recombinants, LMG19668 / p6?« exhibited the highest production of both 3- HPA and 1,3-PD (Figure 3a). Therefore, LMG 19668 / pduF was selected for the subsequent experiments. The duration of biotransformation is a critical factor for industrialization. Consequently, production of 3-HPA and 1,3-PD was examined following 1, 5, 10 and 24 h of culture. Levels of both 3-HPA and 1,3-PD in the media peaked at 5 h in LMG19668 / pc / MF, with levels of 31.3 g / L and 13.0 g / L respectively (Figure 3b).

[0126] Development and optimization of Module I: Stepwise metabolic engineering of L. diolivorans for enhanced 1,3-PD production

[0127] Module I development focused on enhancing 1,3-PD production in L. diolivorans LMG19668, a GRAS strain with native but limited 1,3-PD production capacity. The 1,3-PD biosynthesis pathway involves glycerol uptake, conversion to 3-HPA by glycerol dehydratase, and subsequent reduction to 1,3-PD by 1,3-PD oxidorcductasc (Figure 2). Through stepwise metabolic engineering, four strains were constructed: Lac / 1 (overexpressing glycerol transporter), Lac / 2 (co-expressing glycerol transporter and B 12- dependent glycerol dehydratase with its activation factor), Lac / 3 (co-expressing glycerol transporter and B12-independent glycerol dehydratase with its activation factor), and Lac / 4 (co-expressing glycerol transporter, B 12-independent glycerol dehydratase with its activation factor, and 1,3-PD oxidoreductase) (Figure 1). Using 50 g / L glycerol as substrate, whole-cell biotransformation over 24 h showed Lac / 4 achieved the highest 1,3-PD production compared to control strains (wild-type LMG19668 and LMG19668 / EV) (Figure 7a). Further optimization of Lac / 4 through response surface methodology improved 1,3-PD production to 18 g / L, though 2.1 g / L 3-HPA accumulation remained (Figure 7b).

[0128] EXAMPLE 2: L. diolivorans and E. coli co-culture further enhances 1,3-PD production

[0129] Expression of dhaT (1,3-propanediol oxidoreductase) in E. coli

[0130] The dhaT genes (Genebank No.: WP035771780.1, KRL65223.1, AAM54730.1, WP_003441638.1, WP_260222463.1, WP_032937336.1) encoding 1,3-propanediol oxidoreductase (PDOR) from different microorganisms were ordered from T wist and cloned into pET-28a (+), and transformed into E. coli BL21(DE3) for PDOR expression. The recombinant strain E. colitl, E. colitl, E. coli!3, E. colitA, E. colitl, E. eolith were induced with 0.2 mM IPTG at 16°C overnight. The expressed PDOR protein was analyzed by SDS- PAGE. The result indicated that PDOR was expressed successfully with a molecular weight of around 41 kDa (Figure 4).

[0131] Sequential cultivation enhances 1,3-PD production

[0132] Increasing the expression of PDOR to remove the accumulated 3-HPA can enhance 1,3-PD production. 31.3 g / L 3-HPA and 13.0 g / L 1,3-PD were accumulated in the media from whole cell biotransformation with LMG19668 / p< « . To improve 1,3-PD production with biotransformation of accumulated 3-HPA to 1,3-PD, engineered E. coli expressing PDOR from Clostridium butyricum was used as whole-cell biocatalyst for a subsequent biotransformation step. Supernatant from the L. diolivorans culture was collected and added to E. coli medium, and the E. coli was cultured for 8 h. Levels of 3-HPA and 1,3-PD in the medium was detected with HPLC (Figure 6). After the two-stage biotransformation, 1,3-PD production reached 16.5 g / L (Figure 5a).

[0133] Co-cultivation enhances 1,3-PD production

[0134] Culture of the L. diolivorans EadA strain expressing the glycerol transporter, glycerol dehydratase and 1,3-PD oxidoreductase still resulted in 2.1 g of 3-HPA accumulating in the medium. To improve conversion of 3-HPA to 1,3-PD, six co-cultivation systems (CS1 to CS6) combining the optimal strain from Module I (L. diolivorans Lac / 4, 5 g / L) and engineered E. coli strains from Module II (£. colitl, E. colitl, E. colitl, E. colUA, E. colitl, or E. eolith) were investigated. Different biomass ratios of L. diolivorans and E. coli were cultured in the presence of 50 g / L glycerol (Figure 8). Among all co-cultivation combinations, CS4 consistently demonstrated superior performance across various biomass ratios (Figure 9a-c). The optimized co-cultivation system (1: 1 biomass ratio) achieved 28.03 g / L 1,3-PD production with minimal 3-HPA accumulation (0.2 g / L) (Figure 9d).

[0135] Discussion

[0136] It is desirable to produce high-value platform chemicals more sustainably from waste streams. Both 3-HPA and 1,3-PD are significant platform chemicals which can be derived from glycerol in biodiesel waste. L. diolivorans is advantageous for bioconversion due to its generally regarded as safe (GRAS) status and high robustness in handling the stresses encountered in industrial bioprocesses. However, L. diolivorans has limited 1,3-PD production capacity, and glycerol bioconversion in L. diolivorans is often bottlenecked by the accumulation of 3-HPA, mostly due to insufficient expression of PDOR (DhaT). Herein, it is shown that this bottleneck can be overcome by designing two modules for 3-HPA and 1,3-PD production, respectively, and by reducing 3-HPA accumulation when the two modules arc cultivated sequentially or together (Figure 2 and Figure 8).

[0137] For the engineering of the first module, glycerol transporter derived from E. coli, B. subtilis and L. diolivorans were overexpressed in L. diolivorans LMG19668 to enhance 3-HPA production. With the recombinant LMG19668 / pc?MF as whole-cell biocatalyst, production of 3-HPA was increased.

[0138] The engineering of module I L. diolivorans generated four strains through stepwise overexpression: Lac / 1 (overexpressing glycerol transporter), Lac / 2 (overexpressing glycerol transporter and B 12-dependent glycerol dehydratase with its activation factor), Lac / 3 (overexpressing glycerol transporter and B12-independent glycerol dehydratase with its activation factor), and Lac / 4 (overexpressing glycerol transporter, B 12-independent glycerol dehydratase with its activation factor, and 1,3-PD oxidoreductase).

[0139] For engineering the second module, PDOR from different microorganisms were overexpressed in E. coli), generating strains E. coli / 1-6. These strains were used as wholecell biocatalyst to increase conversion of 3-HPA to 1,3-PD. The co-cultivation systems were established by combining Module T Lac / 4 (5 g / L) with Module TI E. coli strains at various ratios. Among these, co-cultivation system4 (CS4) exhibited optimal performance, particularly at a 1:1 strain ratio, achieving 28.03 g / L 1,3-PD production with minimal 3- HPA accumulation (0.2 g / L).

[0140] The cells of both engineered L. diolivorans and E. coli can be reused for another round with similar production of 3-HPA and 1.3-PD.

[0141] The modular engineering approach using recombinants of L. diolivorans and E. coli has shown improved biocatalysis efficiency, leading to enhanced 3-HPA and 1,3-PD yield. Table 1

Claims

CLAIMS1. A method of preparing 1,3-propanediol from glycerol, the method comprising: a) cultivating Lentilactobacillus diolivorans cells in a cell culture in the presence of glycerol to produce 3-hydroxypropionaldehyde (3-HPA) and / or 1,3-propanediol (1,3-PD), wherein the Lentilactobacillus diolivorans cells are genetically engineered to overexpress a glycerol transporter; and b) cultivating Escherichia coli cells in cell culture media from step a) to produce 1,3- propanediol, wherein the Escherichia coli cells are genetically engineered to overexpress a 1,3-propanediol oxidoreductase.

2. A method of preparing 1,3-propanediol from glycerol, the method comprising cultivating Lentilactobacillus diolivorans cells and Escherichia coli cells in the presence of glycerol, wherein the L. diolivorans is genetically engineered to overexpress a glycerol transporter, and wherein the E. coli is genetically engineered to overexpress a 1,3-propanediol oxidoreductase.

3. The method of claim 1 or 2, wherein the L. diolivorans is L. diolivorans LMG19668 or L. diolivorans DSM14421.

4. The method of any one of claims 1 to 3, wherein the glycerol transporter is a bacterial glycerol transporter.

5. The method of any one of claims 1 to 4, wherein the glycerol transporter is derived from Lentilactobacillus diolivorans.

6. The method of claim 5, wherein the glycerol transporter is derived from L. diolivorans DSM 14421 or LMG19668.

7. The method of claim 6, wherein the glycerol transporter comprises an amino acid sequence having at least 70% sequence identity to an amino acid sequence in SEQ ID8. The method of any one of claims 1 to 7, wherein overexpression of the glycerol transporter increases uptake of glycerol into the genetically engineered L. diolivorans cells compared to wild-type L. diolivorans cells.

9. The method of any one of claims 1 to 8, wherein the L. diolivorans cells are further engineered to overexpress (i) a glycerol dehydratase and a glycerol dehydratase activation factor, and / or (ii) a 1,3-propanediol oxidoreductase.

10. The method of claim 9, wherein the glycerol dehydratase is a bacterial B12-dependent glycerol dehydratase.

11. The method of claim 10, wherein the B12-dependent glycerol dehydratase is derived from Lentilactobacillus diolivorans.

12. The method of claim 11, wherein the B12-dcpcndcnt glycerol dehydratase comprises a polypeptide comprising an amino acid sequence having at least 70% sequence identity to an amino acid sequence in SEQ ID NO: 3, 5 or 7.

13. The method of claim 9, wherein the glycerol dehydratase is a bacterial B 12-independent glycerol dehydratase.

14. The method of claim 13, wherein the B 12-indcpcndcnt glycerol dehydratase is derived from Clostridium butyricum.

15. The method of claim 14, wherein the B 12-independent glycerol dehydratase comprises a polypeptide comprising an amino acid sequence having at least 70% sequence identity to an amino acid sequence in SEQ ID NO: 13.

16. The method of any one of claims 9 to 15, wherein the glycerol dehydratase activation factor comprises a polypeptide comprising an amino acid sequence having at least 70% sequence identity to an amino acid sequence in SEQ ID NO: 9, 11 or 15.

17. The method of any one of claims 9 to 16, wherein overexpression of the glycerol dehydratase increases production of 3-HPA by the genetically engineered L. diolivorans cells compared to wild-type L. diolivorans cells.

18. The method of any one of claims 9 to 17, wherein the 1,3-PD oxidoreductase overexpressed by the L. diolivorans and / or the £’. coli is a bacterial 1,3-propanediol oxidoreductase.

19. The method of claim 18, wherein the 1,3-PD oxidoreductase is derived from Lentilactobacillus diolivorans, Clostridium butyricum, Clostridium pasteurianum, Klebsiella pneumoniae, or Citrobacter freundii.

20. The method of claim 19, wherein the 1,3-PD oxidoreductase comprises an amino acid sequence having at least 70% sequence identity to an amino acid sequence in SEQ ID NO: 17, 19, 21, 23, 25 or 27.

21. The method of any one of claims 1 to 20, wherein overexpression of the 1,3-PD oxidoreductase increases production of 1 ,3-PD by the genetically engineered L. diolivorans or E. coli cells compared to wild-type L. diolivorans or E. coli cells.

22. The method of claim 1 or any one of claims 3 to 21, wherein the method comprises cultivating the Lentilactobacillus diolivorans cells in step a) for at least 3 hours.

23. The method of claim 1 or any one of claims 3 to 22, wherein the method comprises cultivating the E. coli cell in step b) for at least 6 hours.

24. The method of claim 1 or any one of claims 3 to 23, wherein the method comprises isolating media comprising 3-HPA from the cell culture of L. diolivorans cells prior to step b).

25. The method of claim 1 or any one of claims 3 to 23, wherein steps a) and b) are performed concurrently.

26. The method of claim 1 or any one of claims 3 to 25, wherein the method further comprises isolating 1,3-propanediol from the cell culture of E. coli cells.

27. The method of any one of claims 2 to 21 , wherein the method comprises cultivating the L. diolivorans cells and the E. coli cells in the presence of glycerol for at least 18 hours.

28. The method of any one of claims 2 to 21 or claim 27, wherein the E. coli cells comprise at least about 25% of the cells in the co-culturc.

29. The method of claim 28, wherein the E. coli cells comprise about 50% of the cells in the co-culture.

30. The method of any one of claims 2 to 21 or claims 27 to 29, wherein the method comprises isolating 1,3-propanediol from the culture of L. diolivorans and E. coli.

31. A kit for biological production of 1,3-propanediol from glycerol, the kit comprising Lentilactobacillus diolivorans cells genetically engineered to overexpress a glycerol transporter, and Escherichia coli cells genetically engineered to overexpress a 1 ,3- propanediol oxidoreductase.

32. The kit of claim 31, wherein the L. diolivorans cells are further engineered to overexpress (i) a glycerol dehydratase and a glycerol dehydratase activation factor, and / or (ii) a 1,3-propanediol oxidoreductase.

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