CN116240155A - Recombinant bacterium for producing poly (hydroxy fatty acid-co-lactic acid) and application thereof - Google Patents

Recombinant bacterium for producing poly (hydroxy fatty acid-co-lactic acid) and application thereof Download PDF

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CN116240155A
CN116240155A CN202310120542.1A CN202310120542A CN116240155A CN 116240155 A CN116240155 A CN 116240155A CN 202310120542 A CN202310120542 A CN 202310120542A CN 116240155 A CN116240155 A CN 116240155A
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acid
gene
encoding gene
recombinant bacterium
pha
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陈国强
陈江楠
刘絮
张旭
何宏韬
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Beijing Micro Structure Factory Biotechnology Co ltd
Tsinghua University
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Beijing Micro Structure Factory Biotechnology Co ltd
Tsinghua University
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Abstract

The invention provides a recombinant bacterium for producing poly (hydroxy fatty acid-co-lactic acid) and application thereof, wherein the recombinant bacterium capable of producing P (HA-co-LA) is constructed by modifying a bacterial strain which cannot produce P (HA-co-LA), and meanwhile, the recombinant bacterium can regulate and control the content of LA and the content of HA monomers in the produced P (HA-co-LA) to obtain new P (HA-co-LA) materials with excellent performances of various types.

Description

Recombinant bacterium for producing poly (hydroxy fatty acid-co-lactic acid) and application thereof
Technical Field
The invention relates to the technical field of biotechnology and biological materials, in particular to a method for constructing a plurality of hydroxy fatty acid and lactic acid copolymers P (HA-co-LA) and microorganism anabolism pathways and regulating and controlling different monomer proportions.
Background
Polylactic acid (PLA) is a typical type of bio-polyester, and the traditional synthetic means include two steps of bio-fermentation and chemical polymerization, i.e. lactic acid obtained by microbial fermentation, and PLA is formed by catalyst-mediated chemical catalytic polymerization (reference: namoothia, k.m., nair, n.r., and John, r.p. (2010) An overview of the recent developments in Polylactide (PLA) research technical n 101, 8493-8501). PLA is widely applied to the industries of biological medicines, foods and even daily necessities, but has the defects of poor ductility of materials, heavy metal residues and the like, and greatly limits the application of the PLA. Polyhydroxyalkanoates (PHAs) are a class of carbon and energy sources synthesized by microorganisms and stored in cells for aggregation, and are also a class of polymers collectively known as hundreds of monomers. PHA has the characteristics of being renewable and biodegradable. Due to The variety of monomer structures in which PHAs are synthesized, the different types of PHA materials are very diverse in their chemical properties (ref: chen, G.Q., and Hajnal, I. (2015) The 'PHAome' Trends Biotechnol, 559-564). PHA obtained by polymerizing one monomer, such as PHB, has the problems of low elongation at break, brittle material performance, poor toughness and unstable melting state. PLA has a lower melting point than PHB, and significantly reduces the processing difficulty for thermoplastic processes (reference: perego, G., cella, G.D., and Bastoli, C. (1996). Effect of molecular weight and crystallinity on poly (lactic acid) mechanical properties.J Appl Polym Sci 59,37-43.). PHA materials containing lactic acid monomers have a good balance of stiffness and toughness due to the variety of monomers of different structures, and have good degradability and heat sealability. Thus, copolymerizing Lactic Acid (LA) with different types of PHA monomer molecules may improve the performance of PLA or PHA.
In addition, the high cost is an important obstacle for limiting the competition of the biofermentation technology with the traditional chemical technology for market share (reference: taguchi, S., yamada, M., matsumoto, K.i., tajima, K., satoh, Y., munekata, M., ohno, K., kohda, K., shimamura, T., and Kambe, H. (2008) A microbial factory for lactate-based polyesters using a lactate-polymenzyme. Proc Natl Acad Sci 105,17323-1), and the high-density fermentation technology improves the yield per unit volume while increasing the fermentation volume, reduces the fermentation cost, and has obvious application significance.
Disclosure of Invention
In order to solve the problems, the application provides a novel method for preparing P (HA-co-LA) by adopting a biological fermentation technology, and particularly discloses a recombinant bacterium which can produce lactic acid monomers and can copolymerize lactic acid and monomers forming PHA to form P (HA-co-LA) by modifying strains which cannot produce P (HA-co-LA), wherein the recombinant bacterium can regulate and control the LA content and the HA monomer content in the produced P (HA-co-LA), and the obtained P (HA-co-LA) HAs higher glass transition temperature, lower Young modulus and larger elongation at break.
In a first aspect of the invention, there is provided a recombinant bacterium for producing poly (hydroxy fatty acid-co-lactic acid).
The genome of the recombinant bacterium contains PHA synthetic genes and/or lactic acid synthetic genes. Preferably, the PHA synthesis gene and/or lactic acid synthesis gene can be integrated into the genome of the chromosome or can be contained in the recombinant bacterium by being isolated from the carried recombinant plasmid vector.
The PHA synthesis gene and/or lactic acid synthesis gene are over expressed in recombinant bacteria.
The promoter used for the overexpression can be constitutive or inducible. In one embodiment of the present invention, the constitutive promoter may be a constitutive porin gene porin promoter or a mutant thereof. The constitutive promoter may be a low-strength promoter, a medium-strength promoter or a high-strength promoter. Such as Pporin203, pporin221, pporin 194, pporin 278, pporin 68, pporin 42 or Pporin58 (ref: shen, R.et al. Promoter engineering for enhanced P (3 HB-co-4 HB) production by Halomonas blue genes. ACS synthetic biology 7,1897-1906 (2018)).
In one embodiment of the invention, the inducible promoter may be an IPTG inducible T7 promoter, or a T7-like promoter (ref: weiss, R., knight, T. & Sussman, G.cellular computation and communication using engineered genetic regulatory networks.cellular computer, 120-121 (2001)).
The hydroxy fatty acid comprises one or more of 3-hydroxybutyric acid (3 HB), 4-hydroxybutyric acid (4 HB), 3-hydroxypropionic acid (3 HP), 3-hydroxycaproic acid (3-HHx), 6-hydroxycaproic acid (6-HHx), 3-hydroxyoctanoic acid (3 HO), 3-hydroxyvaleric acid (3 HV), 5-hydroxyvaleric acid (5 HV), 3-hydroxydecanoic acid (3 HD), 3-hydroxydodecanoic acid (3 HDD) or 3-hydroxytetradecanoic acid (3 HTD).
The PHA is a homopolymer or a copolymer of monomers. The monomer comprises one or more than two of 3-hydroxybutyric acid (3 HB), 4-hydroxybutyric acid (4 HB), 3-hydroxypropionic acid (3 HP), 3-hydroxycaproic acid (3-HHx), 6-hydroxycaproic acid (6-HHx), 3-hydroxyoctanoic acid (3 HO), 3-hydroxyvaleric acid (3 HV), 5-hydroxyvaleric acid (5 HV), 3-hydroxydecanoic acid (3 HD), 3-hydroxydodecanoic acid (3 HDD) or 3-hydroxytetradecanoic acid (3 HTD).
The PHA synthetic genes include poly (3-hydroxybutyrate) (PHB) synthetic genes, 3-hydroxybutyrate (3 HB) and 4-hydroxybutyrate (4 HB) copolymer (P34 HB) synthetic genes, poly (3-hydroxypropionate) (P3 HP) synthetic genes, etc.
In one embodiment of the invention, the PHA synthesis gene comprising 3-hydroxybutyric acid monomer comprises one or a combination of two or more of a beta-ketothiolase encoding gene (phaA), an acetoacetyl-CoA reductase encoding gene (phaB), or a PHA polymerase encoding gene (phaC).
Preferably, the beta-ketothiolase encoding gene (phaA) and/or the acetoacetyl-CoA reductase encoding gene (phaB) are derived from Eutrophic bacteria.
In one embodiment of the present invention, the PHA synthesis gene comprising the 4-hydroxybutyrate monomer comprises one or a combination of two or more of the succinic semialdehyde dehydrogenase encoding gene (sucD), the 4-hydroxybutyrate dehydrogenase encoding gene (4 hbD), or the 4-hydroxybutyryl-CoA transferase encoding gene (orfZ), or the PHA synthesis gene comprising the 4-hydroxybutyrate monomer comprises one or a combination of two or more of the alcohol dehydrogenase encoding gene (dhaT), the aldehyde dehydrogenase encoding gene (aldD), or the 4-hydroxybutyryl-CoA transferase encoding gene (orfZ). Preferably, one or a combination of two or more of 2-ketoglutarate decarboxylase (ogdA), scpA or scpB is also included.
In one embodiment of the invention, the PHA synthesis genes comprising 3-hydroxypropionic acid monomers comprise an aldehyde dehydrogenase encoding gene (aldD) and/or an alcohol dehydrogenase encoding gene (dhaT).
In one embodiment of the invention, the PHA synthesis gene comprising 3-hydroxydodecanoic acid monomer comprises one or a combination of two or more of a beta-ketothiolase encoding gene (phaA), an acetoacetyl-CoA reductase encoding gene (phaB), or a PHA polymerase encoding gene (phaC), said PHA polymerase encoding gene being phaC61-3.
Wherein the sequence of phaC61-3 is FJ626663.1 at positions 1-1680 or a mutant sequence thereof, such as S325T and Q481K mutation, and in one embodiment of the invention, the sequence is shown in SEQ ID NO:10.
when the hydroxy fatty acid contains 3-hydroxy dodecanoic acid, the recombinant strain is attenuated to express one or more than two of 3-ketoester acyl-CoA thiolase encoding gene (fadA), (S) -3-hydroxy ester acyl-CoA dehydrogenase encoding gene (fadB) or 3-hydroxy acyl-CoA acyltransferase encoding gene (phaG).
Preferably, the attenuation expression may be gene knockout or gene silencing.
Preferably, the recombinant bacterium is attenuated by one or more than two of sdhE, gabD, sad, prpC, phaG or phaJ.
In one embodiment of the present invention, the poly (hydroxy fatty acid-co-lactic acid) has a hydroxy fatty acid of 3HB: the PHA synthesis gene comprises one or a combination of more than two of beta-ketothiolase encoding gene (phaA), acetoacetyl-CoA reductase encoding gene (phaB) or PHA polymerase encoding gene (phaC). Preferably, the beta-ketothiolase encoding gene (phaA) and/or the acetoacetyl-CoA reductase encoding gene (phaB) are derived from Eutrophic bacteria; the recombinant bacteria are Halomonas (Halomonas).
In one embodiment of the present invention, the poly (hydroxy fatty acid-co-lactic acid) has a hydroxy fatty acid of 4HB: the PHA synthesis gene comprises one or more of succinic semialdehyde dehydrogenase encoding gene (sucD), 4-hydroxybutyrate dehydrogenase encoding gene (4 hbD) or 4-hydroxybutyryl-CoA transferase encoding gene (orfZ), or comprises one or more of alcohol dehydrogenase encoding gene (dhaT), aldehyde dehydrogenase encoding gene (aldD) or 4-hydroxybutyryl-CoA transferase encoding gene (orfZ); the recombinant bacteria are Halomonas (Halomonas).
In one embodiment of the present invention, the poly (hydroxy fatty acid-co-lactic acid) has hydroxy fatty acids 3HB and 4HB: the PHA synthesis gene comprises one or a combination of more than two of beta-ketothiolase encoding gene (phaA), acetoacetyl-CoA reductase encoding gene (phaB) or PHA polymerase encoding gene (phaC). Preferably, the beta-ketothiolase encoding gene (phaA) and/or the acetoacetyl-CoA reductase encoding gene (phaB) are derived from Eutrophic bacteria; and a combination comprising one or more of a succinic semialdehyde dehydrogenase encoding gene (sucD), a 4-hydroxybutyrate dehydrogenase encoding gene (4 hbD), or a 4-hydroxybutyryl-coa transferase encoding gene (orfZ), or a combination comprising one or more of an alcohol dehydrogenase encoding gene (dhaT), an aldehyde dehydrogenase encoding gene (aldD), or a 4-hydroxybutyryl-coa transferase encoding gene (orfZ); the recombinant bacteria are Halomonas (Halomonas).
In one embodiment of the present invention, the poly (hydroxy fatty acid-co-lactic acid) has a hydroxy fatty acid of 3HB: PHA synthesis genes comprise a beta-ketothiolase encoding gene (phaA) and an acetoacetyl-CoA reductase encoding gene (phaB); the recombinant bacterium is Escherichia coli.
In one embodiment of the present invention, the poly (hydroxy fatty acid-co-lactic acid) has hydroxy fatty acids 3HB and 4HB: PHA synthesis genes include 2-ketoglutarate decarboxylase (ogdA), succinic semialdehyde dehydrogenase encoding gene (sucD), 4-hydroxybutyrate dehydrogenase encoding gene (4 hbD), 4-hydroxybutyryl-CoA transferase encoding gene (orfZ), alcohol dehydrogenase encoding gene (dhaT), aldehyde dehydrogenase encoding gene (aldD); the recombinant bacterium is Burkholderia, preferably Eubacterium rosenbergii (Ralstonia eutropha).
In one embodiment of the present invention, the poly (hydroxy fatty acid-co-lactic acid) HAs 3HB and mcl HA: the PHA synthesis gene comprises one or more than two of beta-ketothiolase coding gene (phaA), acetoacetyl-CoA reductase coding gene (phaB) or PHA polymerase coding gene (phaC 61-3). Preferably, the beta-ketothiolase encoding gene (phaA) and/or the acetoacetyl-CoA reductase encoding gene (phaB) are derived from Eutrophic bacteria; and, in the recombinant bacteria, weakening and expressing 3-ketoacyl-CoA thiolase gene (fadA), (S) -3-hydroxy ester acyl-CoA dehydrogenase gene (fadB) and 3-hydroxy acyl-CoA acylase (phaG); the recombinant bacteria are Pseudomonas.
The lactic acid synthesis genes comprise polylactic acid synthesis genes.
The lactic acid synthesis gene comprises one or more than two of lactic acid dehydrogenase coding gene, propionyl coenzyme A transferase coding gene or PHA polymerase coding gene.
Preferably, the lactate dehydrogenase-encoding gene is ldhA.
Preferably, the propionyl-coa transferase encoding gene is derived from clostridium propionicum; further preferably, the propionyl-coa transferase encoding gene is pct540; the sequence can be seen at positions 485-2059 of AJ276553.1 or SEQ ID NO:8.
preferably, the PHA polymerase encoding gene is derived from Pseudomonas stutzeri; further preferably, the PHA polymerase encoding gene is phaC1437; the sequence can be found in FJ626663.1 at positions 1-1680 or mutated sequences thereof, e.g., mutations in Q481K, S325T, E D and S477G, in one embodiment of the invention the sequence is found in SEQ ID NO:9.
the recombinant bacteria are Halomonas (Halomonas), pseudomonas (Pseudomonas), escherichia coli or Burkholderia (Burkholderia). Wherein said Burkholderia genus (Burkholderia) includes, but is not limited to, eutrophic bacterium (Ralstonia eutropha). The genus Halomonas (Halomonas) includes Halomonas bluephagenesis TD CGMCC No.4353, halomonas campaniensis LS CGMCC No.6593 or Halomonas aydingkolgenesis M1 CGMCC No.19880 and any derivatives thereof, particularly genetically engineered and physicochemical mutagenic.
The PHA synthesis gene and lactic acid synthesis gene are expressed on chromosome or expressed on plasmid or over expressed.
Preferably, the overexpression may also be an up-regulating of an activating transcription factor of the gene of interest.
In a second aspect of the present invention, a method for constructing the recombinant bacterium described above is provided.
The construction method comprises the step of over-expressing PHA synthetic genes and/or lactic acid synthetic genes in recombinant bacteria.
Preferably, the construction method comprises introducing PHA synthesis genes and/or lactic acid synthesis genes into recombinant bacteria.
Preferably, the PHA synthesis gene and/or lactic acid synthesis gene can be integrated into the genome of the chromosome or can be contained in the recombinant bacterium by being isolated from the carried recombinant plasmid vector.
The introduction may be by inserting PHA synthesis genes and/or lactic acid synthesis genes into non-functional regions of the recombinant bacterium.
The introduction is performed by using a plasmid vector carrying PHA synthesis genes and/or lactic acid synthesis genes, the plasmid vector comprising a promoter, which may be constitutive or inducible. In one embodiment of the present invention, the constitutive promoter may be a constitutive porin gene porin promoter and mutants thereof. The constitutive promoter may be a low-strength promoter, a medium-strength promoter or a high-strength promoter. Such as Pporin203, pporin221, pporin 194, pporin 278, pporin 68, pporin 42 or Pporin58 (ref: shen, R.et al. Promoter engineering for enhanced P (3 HB-co-4 HB) production by Halomonas blue genes. ACS synthetic biology 7,1897-1906 (2018)). In one embodiment of the invention, the inducible promoter may be an IPTG inducible T7 promoter, or a T7-like promoter (ref: weiss, R., knight, T. & Sussman, G.cellular computation and communication using engineered genetic regulatory networks.cellular computer, 120-121 (2001)).
The construction method also comprises the step of knocking out one or more than two of 3-ketoester acyl-CoA thiolase coding genes (fadA), (S) -3-hydroxy ester acyl-CoA dehydrogenase coding genes (fadB) or 3-hydroxy acyl-CoA acylase coding genes (phaG).
Wherein, the knockout can be the knockout of all or part of the nucleotide sequence of the target gene, or the knockout of the regulatory element of the target gene or the related regulatory gene; so that the target gene is not expressed in recombinant bacteria or the expressed protein has no function.
In a third aspect of the present invention, a method of producing poly (hydroxy fatty acid-co-lactic acid) is provided.
The method is an intracellular synthesis method. Preferably, the method comprises fermenting the recombinant bacterium described above.
The fermentation medium may be liquid, solid or semi-solid.
The fermentation medium may be a natural medium, a synthetic medium, and/or a semi-synthetic medium. Preferably, the fermentation medium can be a medium conventional in the prior art, and other substances containing nutritional ingredients can also be used. The conventional medium includes inorganic salt medium (MMG), luria-Bertani medium (LB), and the like.
Preferably, the fermentation medium contains a carbon source, a nitrogen source, an inorganic salt, and the like. The nitrogen source comprises an inorganic nitrogen source and/or an organic nitrogen source. Further preferably, the fermentation medium further comprises vitamins and/or growth factors.
The fermentation medium comprises relevant carbon sources and/or irrelevant carbon sources.
The related carbon source is selected from one or more than two of lactic acid, glycerol, gamma-butyrolactone, 4-hydroxybutyric acid, 1, 2-butanediol, 1, 3-butanediol, 1, 4-butanediol, 1, 6-hexanediol, 1, 8-octanediol, 1, 10-sunflower glycol, 1, 12-dodecanediol, propionic acid, valeric acid or heptanoic acid or other medium-long chain fatty acids;
the non-relevant carbon source is selected from one or more than two of glucose, sodium gluconate, glycerol, acetic acid, caproic acid, xylose, cellulose, lactose, lactic acid or fructose.
The related carbon source and the unrelated carbon source can be used singly or in combination to adjust the proportion of different monomers in P (HA-co-LA).
Preferably, the promoter strength of a single or several genes is regulated to regulate the ratio of the different monomers in P (HA-co-LA).
Preferably, the feeding time in the fermentation process is adjusted to properly prolong the time of cell growth.
In one embodiment of the invention, the feeding time is 5-36 hours of fermentation culture. Preferably 16-28 hours.
In a fourth aspect of the present invention, there is provided a poly (hydroxy fatty acid-co-lactic acid) obtained by the above method.
Wherein the proportion of lactic acid monomer is any one of 0 to 50%, preferably any one of 1 to 25%, for example 1,2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, etc.
In a fifth aspect of the present invention, there is provided a method for regulating the proportion of monomers in poly (hydroxy fatty acid-co-lactic acid), said method comprising fermenting the recombinant bacterium described above.
Preferably, the monomer ratio in poly (hydroxy fatty acid-co-lactic acid) or gene expression using different promoters can be regulated by regulating the carbon source (e.g., the ratio between related and unrelated carbon sources) or regulating the enzyme strength of key nodes of metabolic pathways.
The adjustment may be a turn-up or turn-down. It is preferable to increase the ratio of the lactic acid monomer.
Preferably, the time of carbon nitrogen ratio switching, namely the addition amount of the feed in the bacterial growth stage and the PHA accumulation stage is controlled. In one embodiment of the invention, the feed time during fermentation is adjusted to properly extend the time for cell growth.
In one embodiment of the invention, the feeding time is 5 to 36 hours, preferably 16 to 28 hours, of the fermentation culture.
In a sixth aspect of the present invention, there is provided a method for increasing the proportion of LA in P (HA-co-LA), said method comprising culturing the recombinant bacterium described above by fermentation.
Preferably, the method comprises adjusting the expression intensity of the over-expressed gene. For example, different promoters are used to regulate gene expression, or the ratio of relevant to non-relevant carbon sources in the medium is adjusted.
The seventh aspect of the invention provides an application of the recombinant bacterium and the poly (hydroxy fatty acid-co-lactic acid) in preparing a degradable biological new material. Preferably in the development of medical devices, medical microspheres, surgical sutures, patches, disposable packaging materials or textile fibers, and the like.
The expression of the gene is up-regulated, and the expression is higher than the natural expression level or the expression is changed from the non-expression to the expression.
The expression attenuation is to lower the expression of the gene, lower than the natural expression amount and even not to express.
The poly (hydroxy fatty acid-co-lactic acid) of the present invention is a copolymer of hydroxy fatty acid and lactic acid, which may be abbreviated herein as P (HA-co-LA) or P (LA-co-HA), wherein LA represents lactic acid and HA represents one or more hydroxy fatty acids such as 3HB, 3HB-co-4HB, etc.
The "medium-long chain 3-hydroxy fatty acid" referred to in the present invention is abbreviated as mcl HA, and represents a 3-hydroxy fatty acid having a chain length of 10-15 (e.g., 10, 11, 12, 13, 14, 15), such as 3-hydroxydecanoic acid (3 HD), 3-hydroxydodecanoic acid (3 HDD), 3-hydroxytetradecanoic acid (3 HTD), or the like.
The "proportion of monomer" as used herein is the molar amount of the monomer to the total polymer, for example, the "proportion of monomer in P (3 HB-co-4 HB-co-LA)" as used herein represents the molar amount of monomer LA, 3HB or 4HB to P (3 HB-co-4 HB-co-LA).
The shorthand of the monomer or the polymer and the comparison of Chinese names are shown in the table 1.
Table 1: monomer or polymer shorthand and chinese names
Figure BDA0004079844310000091
Figure BDA0004079844310000101
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The shorthand of the genes and the Chinese names are compared in the application are shown in Table 2.
Table 2: gene shorthand and Chinese name contrast
Figure BDA0004079844310000102
Figure BDA0004079844310000111
Drawings
Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:
fig. 1: p (3 HB-co-LA) anabolism pathway map.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The medium formulation used in the examples:
60LB:5g/L yeast extract, 10g/L peptone, 60g/L NaCl, and the balance distilled water; adjusting the pH value to 7.0-7.2; then autoclaving.
LB:5g/L yeast extract, 10g/L peptone, 10g/L NaCl, and distilled water as the rest; adjusting the pH value to 7.0-7.2; then autoclaving.
20MMG: preparing NaCl solution of yeast extract, wherein the concentration of the yeast extract is 1g/L, and the concentration of NaCl is 20g/L; sterilizing under high pressure after dissolution; after cooling, 1mL of component I was added to each 50mL of solution (to 10g (NH) 4 ) 2 SO 4 And 2g MgSO 4 Distilled water was added to a volume of 200mL, followed by autoclaving) and 1mL of component II (to 96.5g Na 2 HPO 4 ·12H 2 O and 15gKH 2 PO 4 Distilled water is added to fix the volume to 200mL, and then high-pressure steam sterilization is carried out; finally, the pH of the system was adjusted to about 7.0 with 5M aqueous NaOH.
60MMG: preparing NaCl solution of yeast extract, wherein the concentration of the yeast extract is 1g/L, and the concentration of NaCl is 60g/L; sterilizing under high pressure after dissolution; after cooling, 1mL of component I was added to each 50mL of solution (to 10g (NH) 4 ) 2 SO 4 And 2g MgSO 4 Distilled water is added to constant volume to 200mL, and then high-pressure steam sterilization is carried out) And 1mL of component II (96.5 g Na 2 HPO 4 ·12H 2 O and 15gKH 2 PO 4 Distilled water is added to fix the volume to 200mL, and then high-pressure steam sterilization is carried out; finally, the pH of the system was adjusted to about 9.0 with 5M aqueous NaOH.
The dry cell weight calculation method in the examples:
measured as dry cell weight per liter of post-fermentation system. The dry weight of the cells is expressed in g/L. And (5) taking 30mL of bacterial liquid sample in the centrifuge tube, centrifuging to remove the supernatant, and freeze-drying. Cell Dry Weight (CDW) = (weight of centrifuge tube after freeze-drying-weight of raw empty centrifuge tube)/(0.03); the weight of the centrifugal tube after freeze drying and the weight of the original empty centrifugal tube are both in g;0.03 represents 0.03L.
In the examples, the PHA content of the bacterial cells and the respective monomer content were measured by the following methods:
the freeze-dried product is subjected to esterification reaction, and then the monomer content is measured by Gas Chromatography (GC);
esterification reaction: taking 30-40mg of freeze-dried product, placing in an esterification pipe, adding 2mL of chloroform and 2mL of esterification liquid (methanol solution containing 1g/L benzoic acid and 3% concentrated sulfuric acid), uniformly mixing, adding a cover, sealing, and esterifying in a metal bath at 100 ℃ for 4 hours; cooling to room temperature, adding 1mL distilled water, fully oscillating, uniformly mixing, standing and layering; after the chloroform phase is completely separated from water, taking the chloroform phase for gas chromatographic analysis;
taking 20-25mg of poly 3-hydroxybutyrate (PHB), gamma-butyrolactone or delta-valerolactone and D-lactic acid for esterification reaction to serve as a standard sample;
gas Chromatography (GC) analysis parameters: separating the tested substances in an Shimadzu GC-2014 type gas chromatograph by using an HP-5 type chromatographic column; setting a GC analysis temperature rise program, a sample inlet temperature (240 ℃), a detector temperature (250 ℃), an initial temperature and a maintenance time (80 ℃,1.5 min), a first-stage temperature rise (temperature rise rate of 30 ℃/min), a second-stage temperature rise (temperature rise rate of 40 ℃/min, and maintenance time of 2min after rising to 240 ℃), and a total program time of 8min;
and (3) calculating the corresponding PHA monomer ratio by reading the internal standard peak area measured by gas chromatography, the PHA monomer methyl ester peak area of the standard sample, the internal standard peak area of the sample and the PHA monomer methyl ester peak area of the sample.
PHA content (%) = (PHA mass + LA mass)/(freeze-dried product mass x 100%;
LA content (mol%) =mole of LA ≡ (mole of pha+mole of LA) ×100%;
the calculation of the content of the remaining PHA monomers is referred to as the LA content.
Reference or extension papers on the sites of halophiles such as G43, G7, etc. in the examples: low cost commercial production of poly (3-hydroxybutyrate-co-4 hydroxybutyrate) (doctor's university of Qinghua treatise She Jianwen, chen Guojiang), or article: stimulus response-based fine-tuning of polyhydroxyalkanoate pathway in Halomonas (Ye JWet al, metabolic Engineering, 2020).
Halomonas bluephagenesis TD01-D1 used in the examples can be referred to the article: tan D, xue Y S, aibaidula G, et al, unsterile and continuous production of polyhydroxybutyrate by Halomonas TD [ J ]. Bioresource Technology:102:biomass, bioenergy, biowatts, conversion Technologies, biotransformations, production Technologies,2011 (17).
Example 1: introducing pct540 and phaC1437 genes into halophilic bacteria to increase the ratio of synthesizing LA by an irrelevant carbon source
In the present invention, propionyl-CoA transferase (Pct 540) and PLA-polymerizing PHA polymerase (PhaC 1437) were introduced into halophil Halomonas bluephagenesis TD-D1 at the G43 site by homologous recombination. In the copolymer, too high a 3HB ratio tends to result in lack of flexibility and ductility in the material properties, and a number of promoters of different intensities were designed to express the pct540 (SEQ ID NO: 8) and phaC1437 (SEQ ID NO: 9) genes, which are capable of converting the glycolytic product, pyruvic acid (Pyruvate), into Lactyl-CoA (Lactoyl-CoA) and synthesizing P (3 HB-co-LA) under the metabolic pathway described in example 1. The related principle is shown in figure 1.
Promoters in the porin promoter (Pporin) library used included Pporin203, pporin221, pporin 194, pporin 278, pporin 68, pporin 42, pporin58, corresponding recombinant bacteria named Halomonas bluephagenesis TDJ, TDJ2, TDJ3, TDJ4, TDJ5, TDJ6, TDJ7, and the like.
The recombinant bacteria and the parent bacteria Halomonas bluephagenesis TD01 were cultured in 60LB medium at 37℃and 200rpm for 12 hours, and then inoculated in 1% to 50mL 60MMG medium containing glucose as a carbon source, and cultured at 37℃and 200rpm for 48 hours. Cells were collected after 48 hours, and the dry cell weight and PHA content were measured, and the results are shown in Table 3 below.
Table 3: PHA synthesized by various Halomonas bluephagenesis recombinant bacteria and monomer LA thereof
Figure BDA0004079844310000131
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Figure BDA0004079844310000141
The starting bacterium Halomonas bluephagenesis TD, when the non-relevant carbon source (glucose) is used as a substrate, the accumulated PHA does not contain the LA component, while the PHA accumulated by the recombinant bacterium Halomonas bluephagenesis TDJ-TDJ 7 detects the LA component, and 2 genes contained in the strain can realize the synthesis of P (3 HB-co-LA) by taking the non-relevant carbon source as the substrate in the halomonas.
Example 2: introducing phaA, phaB, phaC gene into halophilic bacteria to increase the integral PHA content
According to the invention, exogenous beta-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHA polymerase (phaC) are introduced into a genome at a G51 site by using a Pporin58 promoter and a promoter of a gene cluster of phaCAB from Ralstonia eutropha in halophiles Halomonas bluephagenesis TDJ7 to promote synthesis and copolymerization of 3HB and LA components, so that the yield of P (3 HB-co-LA) is improved. The related principle is shown in figure 1. The strain constructed in this procedure was designated as TDJ7N1.
The recombinant bacterium and the parent bacterium Halomonas bluephagenesis TDJ7 are both cultured in 60LB medium at 200rpm and 37 ℃ for 12 hours, and then inoculated into 50mL 60MMG medium at 1%, and cultured at 200rpm and 37 ℃ for 48 hours. Cells were collected after 48 hours, and the dry cell weight and PHA content were measured, and the results are shown in Table 4 below.
Table 4: expression of phaA, phaB and phaC increases P (LA-co-3 HB) production
Figure BDA0004079844310000151
Under the same culture condition, the recombinant bacterium Halomonas bluephagenesis TDJ N1 has obviously improved cell dry weight and PHA content compared with the starting bacterium Halomonas bluephagenesis TDJ, which shows that the P (3 HB-co-LA) yield of the halomonas can be improved by introducing the 3 genes.
Example 3: introducing a 4HB monomer synthesis pathway into halophiles to increase the proportion of non-related carbon sources to synthesize 4HB
P34HB is a dibasic polyester, and P34HB has better ductility than PHB composed of 3HB alone, and Young's modulus increases with increasing 4HB ratio, so in producing P34HB, increasing 4HB ratio is of great importance in production. The synthetic route of 4HB monomer is divided into two types depending on its source, one is the synthetic route of succinyl-CoA to 4HB consisting of three genes of sucD, 4hbD and orfZ, by which the production of 4HB components using glucose as a substrate can be achieved. The other is the synthetic pathway of BDO to 4HB consisting of three genes of aldD, dhaT, orfZ.
First, two recombinant expression plasmids of different 4HB synthetic pathway genes were constructed, one was pSEVA321-P p2 SucD-4hbD, the other pSEVA321-P p2 AldD-dhaT. Simultaneously, two control plasmids pSEVA321-P are constructed Porin SucD-4hbD, the other pSEVA321-P Porin AldD-dhaT. The four plasmids are respectively transformed into recombinant P at G7 locus of genome Porin H.blue genes TD01G7 of orfZ:: P Porin In orfZ, P34HB synthetic strains were obtained with glucose, BDO or a mixture of both as substrates, respectively. Each strain was subjected to a shake flask fermentation experiment, and the dry weight, PHA% and 4HB% of each strain were analyzed to find a pSEVA 321-P-containing strain p2 SucD-4hbD and pSEVA321-P p2 Strains of aldD-dhaT showed little change in% PHA compared to the control group, but increased to a different extent in% 4HB, see in particular tables 5-6.
Table 5: phaP2 promoter drives succinyl-CoA to 4HB synthetic pathway gene to increase 4HB ratio in P34HB
Figure BDA0004079844310000161
Table 6: phaP2 promoter drives BDO to 4HB synthetic pathway gene to increase 4HB ratio in strain P34HB
Figure BDA0004079844310000162
Example 4: construction of halophilic bacteria for synthesizing P (3 HB-co-4 HB-co-LA)
Recombinant strain of example 2 was introduced into BDO plasmid of example 3, TDJ7N2 (corresponding to P porin -orfZ(P P2 -aldD-dhaT)) and TDJ7N3 (corresponding to P) porin -orfZ(P Porin -aldD-dhaT)). The recombinant bacteria and the parent bacteria Halomonas bluephagenesis TD01 were cultured in 60LB medium at 37℃and 200rpm for 12 hours, and then inoculated at 1% to 50mL of 60MMG medium supplemented with 1, 4-butanediol and glucose as carbon sources, at 37℃and 200rpm for 48 hours. Cells were collected after 48 hours, and the dry cell weight and PHA content were measured, and the results are shown in Table 7 below.
Table 7: PHA synthesized by various Halomonas bluephagenesis recombinant bacteria and conditions of monomers 4HB and LA thereof
Figure BDA0004079844310000163
Figure BDA0004079844310000171
The simultaneous detection of 4HB and LA components in PHA accumulated by recombinant bacterium Halomonas bluephagenesis TDJ N2-J7N3 suggests that microbial production of polyhydroxyfatty acid copolymers containing lactic acid monomers, such as P (3 HB-co-4 HB-co-LA), can be achieved.
Example 5: synthesis of P (3 HB-co-LA) by introducing 3HB and LA monomer Synthesis pathways into E.coli
The invention inserts coenzyme A transferase gene (pct 540, SEQ ID NO: 8) from clostridium propionicum and PHA polymerase gene (phaC 1437, SEQ ID NO: 9) from pseudomonas stutzeri into nonfunctional area gtttctgcgttgtccatacc (SEQ ID NO: 1) site by utilizing CRISPR/Cas9 technology on Escherichia coli genome; inserting beta-ketothiolase gene (phaA) from Eutrophic Roche at gttcctttcattcaatcctc (SEQ ID NO: 2) Re ) acetoacetyl-CoA reductase gene (phaB) Re ) So that the recombinant ECBA can synthesize P (3 HB-co-LA).
The recombinant strain ECBA was cultured in LB medium at 200rpm and 37℃for 12 hours, and then inoculated at 1% to 50mL of MMG medium at 200rmp and 37℃for 48 hours. Carbon sources are respectively added into MMG culture medium: 20g/L glucose, 5g/L lactic acid. Cells were collected after 48 hours, and the dry cell weight and PHA content were measured, and the results are shown in Table 8 below.
Table 8: synthesis of P (3 HB-co-LA) material by recombinant bacterium ECBA in MMG culture medium with different carbon sources
Carbon source Cell dry weight (g/L) PHA content (%) LA(mol％)
Glucose 8.41±1.35 64.50±2.15 12.04±0.65
Glucose + lactic acid 6.68±0.84 52.61±1.97 22.70±1.52
Example 6: synthesis of P (3 HB-co-4 HB-co-LA) by incorporating 3HB, 4HB and LA monomer Synthesis pathways into Eubacterium rosenbergii Ralstonia eutropha
The invention introduces coenzyme A transferase gene (pct 540, SEQ ID NO: 8) from clostridium propionicum and PHA polymerase gene (phaC 1437, SEQ ID NO: 9) from pseudomonas stutzeri into the Ralstonia eutropha of the eubacterium rosenbergii by utilizing a homologous recombination mode at a gaaaatggccactgataaggc (SEQ ID NO: 3) locus of a genome; inserting 2-ketoglutarate decarboxylase (ogdA), succinic semialdehyde dehydrogenase (sucD), 4-hydroxybutyrate dehydrogenase (4 hbd), 4-hydroxybutyryl-CoA transferase (orfZ) at the cttgataaacggaggcatgg (SEQ ID NO: 4) site; an alcohol dehydrogenase (dhaT) and an aldehyde dehydrogenase (aldD) were inserted into the cacagacatttcggcgccgc (SEQ ID NO: 5) site to allow the recombinant REBA to synthesize P (3 HB-co-4 HB-co-LA).
The recombinant REBA was cultured in LB medium at 200rpm and 37℃for 12 hours, and then inoculated at 1% into 50mL of 20MMG medium at 200rmp and 37℃for 48 hours. Carbon sources were added to 20MMG medium: 20g/L fructose, 17g/L palm oil, 2g/L lactic acid. Cells were collected after 48 hours, and the dry cell weight and PHA content were measured, and the results are shown in Table 9 below.
Table 9: synthesis of P (3 HB-co-4 HB-co-LA) material by recombinant bacterium REBA in MMG culture medium with different carbon sources
Figure BDA0004079844310000181
Example 7: synthesis of P (3 HB-co-LA-co-mcl HA) by incorporating 3HB, mcl HA and LA monomer Synthesis pathways into Pseudomonas Pseudomonas entomophila
The invention uses homologous recombination mode to knock out 3-ketoester acyl-CoA thiolase gene (fadA), (S) -3-hydroxy ester acyl-CoA dehydrogenase gene (fadB) and 3-hydroxy acyl-CoA acyl transferase (phaG) on genome in pseudomonas Pseudomonas entomophila. The coenzyme A transferase gene from Clostridium propionicum (pct 540, SEQ ID NO: 8), the PHA polymerase gene from Pseudomonas stutzeri (phaC 1437, SEQ ID NO: 9) were inserted at the ccaccgccagggtaatgacc (SEQ ID NO: 6) locus of the genome; insertion of beta-ketothiolase Gene (phaA) from Eutrophic Ralstonia at the caacggcaccgtgttcggcg (SEQ ID NO: 7) locus Re ) acetoacetyl-CoA reductase gene (phaB) Re ) And PHA polymerase gene (phaC 61-3, seq ID NO:10 To allow the recombinant PEBA to synthesize P (3 HB-co-LA-co-mclHA).
After culturing the recombinant PEBV in LB medium at 200rpm and 30℃for 12 hours, the recombinant PEBV was inoculated at 1% into 50mL MS medium containing 20g/L glucose, 2g/L lauric acid and 2g/L lactic acid, and cultured at 200rpm and 30℃for 72 hours, and then cells were collected, and the cell dry weight and PHA content were measured, and the results were shown in Table 10 below.
Table 10: synthesis of P (3 HB-co-LA-co-mclHA) material by recombinant PEBA in MMG culture medium with different carbon sources
Carbon source Cell dry weight (g/L) PHA content (%) LA(mol％) 3HDD(mol％)
Glucose 6.53±0.75 48.56±1.48 13.57±0.78 0
Lauric acid 3.58±0.88 22.15±3.86 1.64±0.38 87.42±3.19
Glucose+lauric acid 5.95±2.36 62.36±1.46 8.72±1.56 33.67±1.08
Glucose+lauric acid+lactic acid 4.32±0.67 52.71±2.97 14.37±0.96 28.53±3.66
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims (14)

1. A recombinant bacterium for producing poly (hydroxy fatty acid-co-lactic acid), wherein the genome of the recombinant bacterium comprises a PHA synthesis gene and a lactic acid synthesis gene.
2. The recombinant bacterium of claim 1, wherein the hydroxy fatty acid comprises one or more of 3-hydroxybutyric acid, 4-hydroxybutyric acid, 3-hydroxypropionic acid, 3-hydroxycaproic acid, 6-hydroxycaproic acid, 3-hydroxyoctanoic acid, 3-hydroxyvaleric acid, 5-hydroxyvaleric acid, 3-hydroxydecanoic acid, 3-hydroxydodecanoic acid, or 3-hydroxytetradecanoic acid;
the PHA is a homopolymer or a copolymer of monomers; the monomer comprises one or more than two of 3-hydroxybutyric acid, 4-hydroxybutyric acid, 3-hydroxypropionic acid, 3-hydroxycaproic acid, 6-hydroxycaproic acid, 3-hydroxycaprylic acid, 3-hydroxyvaleric acid, 5-hydroxyvaleric acid, 3-hydroxydecanoic acid, 3-hydroxydodecanoic acid or 3-hydroxytetradecanoic acid.
3. The recombinant bacterium according to claim 1 or 2, wherein:
the PHA synthesis gene comprising 3-hydroxybutyric acid monomer comprises one or a combination of more than two of a beta-ketothiolase encoding gene, an acetoacetyl-CoA reductase encoding gene or a PHA polymerase encoding gene;
the PHA synthesis gene comprising 4-hydroxybutyrate monomer comprises one or more of 2-ketoglutarate decarboxylase, succinic semialdehyde dehydrogenase encoding gene, 4-hydroxybutyrate dehydrogenase encoding gene or 4-hydroxybutyryl-CoA transferase encoding gene, or comprises one or more of alcohol dehydrogenase encoding gene, aldehyde dehydrogenase encoding gene or 4-hydroxybutyryl-CoA transferase encoding gene;
PHA synthesis genes comprising 3-hydroxypropionic acid monomers comprise aldehyde dehydrogenase encoding genes and/or alcohol dehydrogenase encoding genes;
the PHA synthesis gene containing 3-hydroxydodecanoic acid monomer contains one or more than two of beta-ketothiolase coding gene, acetoacetyl-CoA reductase coding gene or PHA polymerase coding gene, and the PHA polymerase coding gene is phaC61-3.
4. The recombinant bacterium according to claim 3, wherein when the hydroxy fatty acid comprises 3-hydroxydodecanoic acid, the recombinant bacterium is attenuated to express one or a combination of two or more of a 3-ketoacyl-coa thiolase encoding gene, an (S) -3-hydroxy acyl-coa dehydrogenase encoding gene, or a 3-hydroxyacyl-coa acyltransferase encoding gene.
5. The recombinant bacterium according to claim 1, wherein the lactic acid synthesis gene comprises one or a combination of two or more of a lactic acid dehydrogenase encoding gene, a propionyl-coa transferase encoding gene, or a PHA polymerase encoding gene;
the lactate dehydrogenase encoding gene is ldhA;
the propionyl coenzyme A transferase coding gene is pct540;
the PHA polymerase encoding gene is phaC1437.
6. The recombinant bacterium according to claim 1, wherein the recombinant bacterium is a species of the genus Halomonas (Halomonas), pseudomonas (Pseudomonas), escherichia coli (Escherichia) or Burkholderia (Burkholderia).
7. The recombinant bacterium according to claim 1, wherein the PHA synthesis gene and the lactic acid synthesis gene are expressed on chromosomes or on plasmids.
8. A method of constructing a recombinant bacterium according to any one of claims 1 to 7, wherein the method comprises overexpressing PHA synthesis genes and/or lactic acid synthesis genes in the recombinant bacterium.
9. The method according to claim 8, wherein the method comprises knocking out one or a combination of two or more of a 3-ketoester acyl-coa thiolase encoding gene, an (S) -3-hydroxy ester acyl-coa dehydrogenase encoding gene, and a 3-hydroxy acyl-coa acyltransferase encoding gene.
10. A method for producing poly (hydroxy fatty acid-co-lactic acid), comprising fermenting the recombinant bacterium of any one of claims 1-7.
11. The method of claim 10, wherein the fermentation medium comprises a related carbon source and/or a non-related carbon source;
the related carbon source is selected from one or more than two of lactic acid, glycerol, gamma-butyrolactone, 4-hydroxybutyric acid, 1, 2-butanediol, 1, 3-butanediol, 1, 4-butanediol, 1, 6-hexanediol, 1, 8-octanediol, 1, 10-sunflower glycol, 1, 12-dodecanediol, propionic acid, valeric acid or heptanoic acid;
the non-relevant carbon source is selected from one or more than two of glucose, sodium gluconate, glycerol, acetic acid, caproic acid, xylose, cellulose, lactose, lactic acid or fructose.
12. A poly (hydroxy fatty acid-co-lactic acid) obtained by the method of claim 10 or 11.
13. A method for regulating the proportion of monomers in poly (hydroxy fatty acid-co-lactic acid), comprising fermenting the recombinant bacterium of any one of claims 1-7.
14. Use of the recombinant bacterium of any one of claims 1-7, the poly (hydroxy fatty acid-co-lactic acid) of claim 12, for the preparation of a degradable biological new material.
CN202310120542.1A 2023-02-13 2023-02-13 Recombinant bacterium for producing poly (hydroxy fatty acid-co-lactic acid) and application thereof Pending CN116240155A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115851510A (en) * 2022-10-13 2023-03-28 深圳中科翎碳生物科技有限公司 Halomonas and application thereof in co-production of tetrahydropyrimidine and polyhydroxyalkanoates

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115851510A (en) * 2022-10-13 2023-03-28 深圳中科翎碳生物科技有限公司 Halomonas and application thereof in co-production of tetrahydropyrimidine and polyhydroxyalkanoates
CN115851510B (en) * 2022-10-13 2023-09-12 深圳中科翎碳生物科技有限公司 Salmonella and application thereof in co-production of tetrahydropyrimidine and polyhydroxyalkanoate

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